Oligonucleotide with protected base

ABSTRACT

The present invention provides a protected nucleotide for elongation, which can be purified efficiently and in a high yield by a liquid-liquid extraction operation, and can achieve an oligonucleotide production method by a phosphoramidite method. 
     It has been found that the above-mentioned problem can be solved by a particular oligonucleotide comprising a protected base and/or particular oligonucleotide protected by a branched chain-containing aromatic group at 3′-position.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a particular oligonucleotide comprisinga protected base and a production method of an oligonucleotide using thesame. In addition, the present invention relates to a particularbranched chain-containing aromatic protecting group, an oligonucleotidehaving a 3′-hydroxyl group protected by said protecting group, and aproduction method of an oligonucleotide using the oligonucleotide havinga 3′-hydroxyl group protected by said protecting group.

BACKGROUND OF THE INVENTION

The synthesis method of oligonucleotide includes a phosphate triestermethod, an H-phosphonate method, a phosphoramidite method and the like,and solid phase synthesis (solid phase method) using a phosphoramiditemethod is most widely used at present (non-patent document 1). The solidphase method is advantageous from the aspect of speed, since process hasbeen optimized and automation has progressed. However, it is associatedwith defects in that scaling-up is limited due to facility restriction,reagents and starting materials are used in excess, and confirmation ofthe progress status of the reaction in an intermediate step, analysis ofintermediate structure and the like are difficult.

The synthesis methods of oligonucleotide by a liquid phase method havealso been studied. Generally, however, treatments after each reactionare performed by a method including (1) directly concentrating thereaction mixture, followed by isolation and purification by silica gelcolumn chromatography, (2) extracting with a solvent such as methylenechloride, chloroform and the like, washing with an aqueous solution,concentrating, purifying by silica gel column chromatography, and thelike, and the operation is complicated and the yield is low. Inparticular, a large-scale, rapid synthesis of a long oligonucleotide isdifficult, and the methods are impractical as industrial productionprocesses.

In recent years, a pseudo-solid phase method-like approach has beenreported as an attempt to solve the respective defects of the liquidphase method and the solid phase method, and an oligonucleotideproduction method using a soluble polymer such asmonomethoxypolyethylene glycol (MPEG) and the like as a protecting groupis disclosed as one example thereof (non-patent document 2). However,while synthetic examples of up to 20mer DNA are disclosed, acrystallization isolation operation is essential for each reaction, andthe progress status of the reaction and the like are difficult toconfirm, since MPEG molecule itself is not a unimolecule.

In addition, as a pseudo-solid phase method-like method, a productionmethod of oligonucleotide including use of an ionic liquid as aprotecting group has been reported, and Synthetic Examples of DNA up topentamers are shown (non-patent document 3). However, the method isinferior to a solid phase method in the speed and efficiency, since acrystallization isolation operation is essential for each reaction andan operation time is necessary therefor.

Furthermore, a synthesis method of oligonucleotide comprising use of ahydrophobic group-linked nucleoside is disclosed (patent document 1).While it has been reported that the method affords synthesis of 21meroligonucleotide, it is markedly complicated, since the method requiressolidification isolation in every step of deprotection of 5′-protectinggroup, coupling and oxidation.

DOCUMENT LIST Patent Document

-   patent document 1: JP-A-2010-275254

Non-Patent Documents

-   non-patent document 1: S. L. Beaucage, D. E. Bergstorm, G. D.    Glick, R. A. Jones, Current Protocols in Nucleic Acid Chemistry;    John Wiley & Sons (2000)-   non-patent document 2: Nucleic Acid Res., 1990, Vol. 18, No. 11,    3155-3159-   non-patent document 3: J. Org. Chem., 2006, Vol. 71, No. 20,    7907-7910

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

The problem of the present invention is provision of a protectednucleotide for elongation, which can afford a production method ofoligonucleotide by a phosphoramidite method, which enables efficientpurification in a high yield by a liquid-liquid extraction operation.

Means of Solving the Problems

As a result of the intensive studies, the present inventors have foundthat the above-mentioned problem can be achieved by a particularoligonucleotide comprising a protected base, which resulted in thecompletion of the present invention.

The present invention includes the following.

-   [1] An oligonucleotide comprising a protected base, which is    represented by the formula (I):

-   wherein q is any integer of not less than 0;-   Base² in the number of q+1 are each independently a nucleic acid    base protected by a group having a C₅₋₃₀ straight chain or branched    chain alkyl group and/or a C₅₋₃₀ straight chain or branched chain    alkenyl group;-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    halogen atom or an organic group crosslinked with the 4-position    carbon atom;-   X′ in the number of q are each independently a hydrogen atom, an    optionally protected hydroxyl group, a halogen atom or an organic    group crosslinked with the 4-position carbon atom;-   P² in the number of q+1 are each independently a protecting group    removable under basic conditions;-   R³⁴ in the number of q are each independently an oxygen atom or a    sulfur atom; and-   R_(e) and R_(f) are each independently a C₁₋₆ alkyl group, or a 5-    or 6-membered saturated cyclic amino group formed together with the    adjacent nitrogen atom.-   [2] The oligonucleotide comprising a protected base of [1], wherein    q is 0.-   [3] The oligonucleotide comprising a protected base of [1] or [2],    wherein the group having a C₅₋₃₀ straight chain or branched chain    alkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenyl    group is-   a group represented by the formula (k):

-   wherein * indicates the bonding position to a nucleic acid base;-   R²⁷ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (l):

-   wherein * indicates the bonding position to a nucleic acid base;-   Q₁ is —O—, —S— or —NR³⁰— wherein R³⁰ is a hydrogen atom or a C₁₋₂₂    alkyl group;-   R_(c) and R_(d) are each independently a hydrogen atom or a C₁₋₂₂    alkyl group; and-   R²⁸ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (m):

-   wherein * indicates the bonding position to a nucleic acid base;-   l is an integer of 1 to 5;-   Q₂ in the number of l are each independently a single bond, or —O—,    —S—, —OC(═O)—, —C(═O)O—, —O—CH₂—, —NH—, —NHC(═O)—, —C(═O)NH—,    —NH—CH₂— or —CH₂—;-   R²⁹ in the number of l are each independently a C₅₋₃₀ straight chain    or branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group;-   ring C is a benzene ring or a cyclohexane ring, each optionally    having, in addition to Q₂R²⁹ in the number of l and *C═O, a    substituent selected from the group consisting of a halogen atom, a    C₁₋₆ alkyl group optionally substituted by one or more halogen    atoms, and a C₁₋₆ alkoxy group optionally substituted by one or more    halogen atoms, or-   a group represented by the formula (s):

-   wherein * indicates the position at which an imino bond is formed    with an amino group of a nucleic acid base; and-   R³⁵ and R³⁶ are each independently a C₅₋₃₀ straight chain or    branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group.-   [4] The oligonucleotide comprising a protected base of [3], wherein    R²⁷, R²⁸, R²⁹ in the number of l, R³⁵ and R³⁶ are each independently    a branched chain alkyl group or branched chain alkenyl group    selected from the group consisting of a    2,6,10,14-tetramethylpentadecyl group, a 2,6,10-trimethylundecyl    group, a 2,2,4,8,10,10-hexamethyl-5-undecyl group, a    2,6,10-trimethylundeca-1,5,9-trienyl group, a 2,6-dimethylheptyl    group, a 2,6-dimethylhept-5-enyl group, a    2,6-dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a    12-methyltridecyl group, an 11-methyltridecyl group, an    11-methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl    group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-methyloctyl    group, a 3,7-dimethyloctyl group, a 3-methylheptyl group, a    3-ethylheptyl group, a 5-undecyl group, a 2-heptyl group, a    2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptyl group, a    4-heptyl group, a 4-methyl-pentyl group, a 3-methyl-pentyl group,    and a 2,4,4-trimethylpentyl group; or a straight chain alkyl group    selected from the group consisting of a tetradecyl group, a tridecyl    group, a dodecyl group, an undecyl group, a decyl group, a nonyl    group, an octyl group, a heptyl group, a hexyl group, and a pentyl    group.-   [5] The oligonucleotide comprising a protected base of any one of    [1] to [4], wherein the C₅₋₃₀ straight chain or branched chain alkyl    group and/or C₅₋₃₀ straight chain or branched chain alkenyl group is    a C₅₋₃₀ branched chain alkyl group and/or a C₅₋₃₀ branched chain    alkenyl group.-   [6] The oligonucleotide comprising a protected base of any one of    [1] to [5], wherein P¹ is a monomethoxytrityl group or a    dimethoxytrityl group.-   [7] A method of producing an oligonucleotide, comprising using the    oligonucleotide comprising a protected base of any one of [1] to    [6].-   [8] A method of producing an n+p-mer oligonucleotide, comprising-   (2) a step of condensing a p-mer oligonucleotide comprising a    protected base (p is any integer of one or more) wherein the    3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group is    protected by a temporary protecting group removable under acidic    conditions, and the nucleic acid base is protected by a group having    a C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀    straight chain or branched chain alkenyl group, with an n-mer    oligonucleotide (n is an integer of one or more) wherein the    5′-hydroxyl group is not protected and the 3′-hydroxyl group is    protected, by forming a phosphite triester bond via the 5′-hydroxyl    group thereof.-   [9] The production method of [8], wherein p is 1.-   [10] The production method of [8] or [9], further comprising the    following step (3):-   (3) a step of converting the phosphite triester bond of the n+p-mer    oligonucleotide obtained in the condensation step to a phosphate    triester bond or a thiophosphate triester bond by adding an    oxidizing agent or a sulfurizing agent to the reaction mixture    obtained in the condensation step (2).-   [11] The production method of any one of [8] to [10], further    comprising the following step (1):-   (1) a step of removing the temporary protecting group removable    under acidic conditions of the 5′-hydroxyl group by reacting, in a    non-polar solvent prior to the condensation step (2), an n-mer    oligonucleotide wherein the 3′-hydroxyl group is protected, and the    5′-hydroxyl group is protected by a temporary protecting group, with    an acid.-   [12] The production method of [11], wherein the step (1) is    performed in the presence of at least one kind of cation scavenger    selected from a pyrrole derivative and an indole derivative, and    further comprises a step of neutralization with an organic base    after removal of the temporary protecting group of the 5′-hydroxyl    group.-   [13] The production method of any one of [10] to [12], further    comprising the following step (4):-   (4) a step of isolating the n+p-mer oligonucleotide from the    reaction mixture obtained in step (3) by an extraction operation    alone.-   [14] The method of [13], further comprising the following step (5):-   (5) a step of removing all the protecting groups of the n+p-mer    oligonucleotide obtained in step (4).-   [15] The production method of any one of [8] to [14], wherein the    p-mer oligonucleotide comprising a protected base, wherein the    3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group is    protected by a temporary protecting group removable under acidic    conditions, and the nucleic acid base is protected by a group having    a C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀    straight chain or branched chain alkenyl group, is the    oligonucleotide comprising a protected base of any one of [1] to    [6].-   [16] The production method of any one of [8] to [15], wherein the    3′-hydroxyl group of the n-mer oligonucleotide is protected by a    group represented by the formula (III):    -L-Y—Z  (III)    wherein-   L is a group represented by the formula (a1):

-   wherein * shows the bonding position to Y; ** indicates the bonding    position to a 3′-hydroxy group of the nucleotide;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is a single bond, or a group represented by    **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows the bonding position to    L₁, *** shows the bonding position to C═O, R¹ is an optionally    substituted C₁₋₂₂ alkylene group, and R² and R³ are each    independently a hydrogen atom or an optionally substituted C₁₋₂₂    alkyl group, or-   R² and R³ are optionally joined to form an optionally substituted    C₁₋₂₂ alkylene bond,-   Y is an oxygen atom or NR wherein R is a hydrogen atom, an alkyl    group or an aralkyl group, and-   Z is a group represented by the formula (a2):

-   wherein * shows the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to R⁴, QR⁵ in the number    of k and *C(R_(a))(R_(b)), a substituent selected from the group    consisting of a halogen atom, a C₁₋₆ alkyl group optionally    substituted by one or more halogen atoms, and a C₁₋₆ alkoxy group    optionally substituted by one or more halogen atoms;-   R_(a) is a hydrogen atom; and-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   wherein * indicates a bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are each independently as defined above;-   R⁷ in the number of j are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of j    and R⁶, a substituent selected from the group consisting of a    halogen atom, a C₁₋₆ alkyl group optionally substituted by one or    more halogen atoms, and a C₁₋₆ alkoxy group optionally substituted    by one or more halogen atoms.-   [17] The production method of any one of [8] to [16], wherein at    least one nucleic acid base of the n-mer oligonucleotide is    protected by a group having a C₅₋₃₀ straight chain or branched chain    alkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenyl    group.-   [18] The production method of any one of [8] to [17], wherein the    group having a C₅₋₃₀ straight chain or branched chain alkyl group    and/or a C₅₋₃₀ straight chain or branched chain alkenyl group is a    group represented by the formula (k):

-   wherein * indicates the bonding position to a nucleic acid base; and-   R²⁷ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (l):

-   wherein * indicates the bonding position to a nucleic acid base;-   Q₁ is —O—, —S— or —NR³⁰— wherein R³⁰ is a hydrogen atom or a C₁₋₂₂    alkyl group;-   R_(c) and R_(d) are each independently a hydrogen atom or a C₁₋₂₂    alkyl group; and-   R²⁸ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (m):

-   wherein * indicates the bonding position to a nucleic acid base;-   l is an integer of 1 to 5;-   Q2 in the number of l are each independently a single bond, or —O—,    —S—, —CO(═O)—, —C(═O)O—, —O—CH₂—, —NH—, —NHC(═O)—, —C(═O)NH—,    —NH—CH₂— or —CH₂—;-   R²⁹ in the number of l are each independently a C₅₋₃₀ straight chain    or branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group; and-   ring C is a benzene ring or a cyclohexane ring each optionally    having, in addition to Q₂R²⁹ in the number of l and *C═O, a    substituent selected from the group consisting of a halogen atom, a    C₁₋₆ alkyl group optionally substituted by one or more halogen    atoms, and a C₁₋₆ alkoxy group optionally substituted by one or more    halogen atoms), or-   a group represented by the formula (s):

-   wherein * indicates the position at which an imino bond is formed    with an amino group of a nucleic acid base; and-   R³⁵ and R³⁶ are each independently a C₅₋₃₀ straight chain or    branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group.-   [19] The production method of [18], wherein R²⁷, R²⁸, R²⁹ in the    number of l, R³⁵ and R³⁶ are each independently a branched chain    alkyl group or branched chain alkenyl group selected from the group    consisting of a 2,6,10,14-tetramethylpentadecyl group, a    2,6,10-trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecyl    group, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a    2,6-dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a    2,6-dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a    12-methyltridecyl group, an 11-methyltridecyl group, an    11-methyldodecyl group, a 10-methylundecyl group, an 8-heptadecyl    group, a 7-pentadecyl group, a 7-methyloctyl group, a 3-methyloctyl    group, a 3,7-dimethyloctyl group, a 3-methylheptyl group, a    3-ethylheptyl group, a 5-undecyl group, a 2-heptyl group, a    2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptyl group, a    4-heptyl group, a 4-methyl-pentyl group, a 3-methyl-pentyl group,    and a 2,4,4-trimethylpentyl group; or a straight chain alkyl group    selected from the group consisting of a tetradecyl group, a tridecyl    group, a dodecyl group, an undecyl group, a decyl group, a nonyl    group, an octyl group, a heptyl group, a hexyl group, and a pentyl    group.-   [20] The production method of any one of [8] to [19], wherein the    C₅₋₃₀ straight chain or branched chain alkyl group and/or C₅₋₃₀    straight chain or branched chain alkenyl group are/is a C₅₋₃₀    branched chain alkyl group and/or a C₅₋₃₀ branched chain alkenyl    group.-   [21] A pharmaceutical product comprising the oligonucleotide    produced by the production method of any one of [7] to [20].-   [22] An oligonucleotide protected by a branched chain-containing    aromatic group, which is represented by the formula (II):

wherein

-   m is any integer of 0 or more;-   Base¹ in the number of m+1 are each independently an optionally    protected nucleic acid base;-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    halogen atom or an organic group crosslinked with the 4-position    carbon atom;-   X′ in the number of m are each independently a hydrogen atom, an    optionally protected hydroxyl group, a halogen atom or an organic    group crosslinked with the 4-position carbon atom;-   P² in the number of m are each independently a protecting group    removable under basic conditions;-   R³⁴ in the number of m are each independently an oxygen atom or a    sulfur atom;-   L is a group represented by the formula (a1):

-   wherein * shows the bonding position to Y; ** indicates the bonding    position to a 3′-hydroxy group of the nucleotide;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is a single bond, or a group represented by    **C(═O)N(R²)—R¹—N(R³)*** wherein shows the bonding position to L₁,    *** shows the bonding position to C═O, R¹ is an optionally    substituted C₁₋₂₂ alkylene group, and R² and R³ are each    independently a hydrogen atom or an optionally substituted C₁₋₂₂    alkyl group, or-   R² and R³ are optionally joined to form an optionally substituted    C₁₋₂₂ alkylene bond,-   Y is an oxygen atom or NR wherein R is a hydrogen atom, an alkyl    group or an aralkyl group, and-   Z is a group represented by the formula (a2):

-   wherein * shows the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains, and the total carbon number of not less than 14 and not more    than 300;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to R⁴, QR⁵ in the number    of k and *C(R_(a))(R_(b)), a substituent selected from the group    consisting of a halogen atom, a C₁₋₆ alkyl group optionally    substituted by one or more halogen atoms, and a C₁₋₆ alkoxy group    optionally substituted by one or more halogen atoms;-   R_(a) is a hydrogen atom; and-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   wherein * indicates the bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are each independently as defined above;-   R⁷ in the number of j are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of j    and R⁶, substituent(s) selected from the group consisting of a    halogen atom, a C₁₋₆ alkyl group optionally substituted by one or    more halogen atoms, and a C₁₋₆ alkoxy group optionally substituted    by one or more halogen atoms.-   [23] The oligonucleotide of [22], wherein m is 0.-   [24] The oligonucleotide of [22] or [23], wherein R⁵ and R⁷ are each    independently a 3,7,11,15-tetramethylhexadecyl group, a    3,7,11-trimethyldodecyl group, a    2,2,4,8,10,10-hexamethyl-5-dodecanoyl group, a    3,4,5-tri(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group, or a    3,5-di(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group.-   [25] The oligonucleotide of [22] or [23], wherein -L-Y—Z is selected    from the group consisting of a    2-{2,4-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a 3,5-di(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    4-(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    2-{1-[(2-chloro-5-(2′,3′-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl    group; a 3,4,5-tri(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    2-{3,4,5-tri(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{4-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{2-[3′,4′,5′-tri(2″,3″-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl    group; a    2-{4-(2′,3′-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl    group; a 4-(2′,3′-dihydrophytyloxy)-2-methylbenzylsuccinyl group; a    2-{4-(2′,3′-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl    group; a    4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl group;    a    2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl    group; a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group; a    2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{3,5-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{1-[2,3,4-tri(2′,3′-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl    group; a    2-{1-[4-(2′,3′-dihydrophytyloxy)phenyl]-4′-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylsuccinyl    group; and a    2-{3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl    group.-   [26] The oligonucleotide of any one of [22] to [25], wherein at    least one of the nucleic acid bases is protected by a group having a    C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀    straight chain or branched chain alkenyl group.-   [27] A protecting group of nucleotide 3′-hydroxyl group, which is    represented by the formula (III):    -L-Y—Z  (III)    wherein-   L is a group represented by the formula (a1):

-   wherein * shows the bonding position to Y; ** indicates the bonding    position to a 3′-hydroxy group of the nucleotide;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is a single bond, or a group represented by    **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows the bonding position to    L₁, *** shows the bonding position to C═O, R¹ is an optionally    substituted C₁₋₂₂ alkylene group, and R² and R³ are each    independently a hydrogen atom or an optionally substituted C₁₋₂₂    alkyl group, or-   R² and R³ are optionally joined to form an optionally substituted    C₁₋₂₂ alkylene bond,-   Y is an oxygen atom or NR wherein R is a hydrogen atom, an alkyl    group or an aralkyl group, and-   Z is a group represented by the formula (a2):

-   wherein * shows the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), R⁴ is optionally a single bond or —O— in    combination with R⁶ to form a fluorenyl group or a xanthenyl group    together with ring B;-   Q in the number of k are each independently a single bond, or —O—,    —S—, —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to R⁴, QR⁵ in the number    of k and *C(R_(a))(R_(b)), a substituent selected from the group    consisting of a halogen atom, a C₁₋₆ alkyl group optionally    substituted by one or more halogen atoms, and a C₁₋₆ alkoxy group    optionally substituted by one or more halogen atoms;-   R_(a) is a hydrogen atom; and-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   wherein * indicates the bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are each independently as defined above;-   R⁷ in the number of j are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   R⁶ is a hydrogen atom, or optionally a single bond or —O— in    combination with R⁴ to form a fluorenyl group or a xanthenyl group    together with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of j    and R⁶, a substituent selected from the group consisting of a    halogen atom, a C₁₋₆ alkyl group optionally substituted by one or    more halogen atoms, and a C₁₋₆ alkoxy group optionally substituted    by one or more halogen atoms.-   [28] The protecting group of [27], wherein R⁵ and R⁷ are each    independently a 3,7,11,15-tetramethylhexadecyl group, a    3,7,11-trimethyldodecyl group, a    2,2,4,8,10,10-hexamethyl-5-dodecanoyl group, a    3,4,5-tri(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group, or a    3,5-di(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group.-   [29] The protecting group of [27], which is selected from the group    consisting of a    2-{2,4-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a 3,5-di(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    4-(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    2-{1-[(2-chloro-5-(2′,3′-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl    group; a 3,4,5-tri(2′,3′-dihydrophytyloxy)benzylsuccinyl group; a    2-{3,4,5-tri(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{4-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{2-[3′,4′,5′-tri(2″,3″-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl    group; a    2-{4-(2′,3′-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl    group; a 4-(2′,3′-dihydrophytyloxy)-2-methylbenzylsuccinyl group; a    2-{4-(2′,3′-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl    group; a    4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl group;    a    2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl    group; a 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group; a    2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{3,5-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    2-{1-[2,3,4-tri(2′,3′-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl    group; a    2-{1-[4-(2′,3′-dihydrophytyloxy)phenyl]-4′-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; a    3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylsuccinyl    group; and a    2-{3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl    group.-   [30] A method of producing an n′+p′-mer oligonucleotide comprising:-   (2′) a step of condensing a p′-mer oligonucleotide (p′ is any    integer of one or more) wherein the 3′-hydroxyl group is    phosphoramidited, the 5′-hydroxyl group is protected by a temporary    protecting group removable under acidic conditions, and the nucleic    acid base is optionally protected, with an n′-mer oligonucleotide    (n′ is any integer of one or more) wherein the 5′-hydroxyl group is    not protected, and the 3′-hydroxyl group is protected by the    protecting group according to any one of [27] to [29], by forming a    phosphite triester bond via the 5′-hydroxyl group thereof.-   [31] The production method of [30], wherein p′ is 1.-   [32] The production method of [30] or [31], further comprising the    following step (3′):-   (3′) a step of converting the phosphite triester bond of the    n′+p′-mer oligonucleotide obtained by the condensation step to a    phosphate triester bond or a thiophosphate triester bond by adding    an oxidizing agent or a sulfurizing agent to the reaction mixture    obtained in the condensation step (2′).-   [33] The production method of any one of [30] to [32], further    comprising the following step (1′):-   (1′) a step of removing a temporary protecting group removable under    acidic conditions of the 5′-hydroxyl group by reacting, in a    non-polar solvent prior to the condensation step (2′), the n′-mer    oligonucleotide wherein the 3′-hydroxyl group is protected by the    protecting group according to any one of [27] to [29], and the    5′-hydroxyl group is protected by the temporary protecting group,    with an acid.-   [34] The production method of [33], wherein step (1′) is performed    in the presence of at least one kind of cation scavenger selected    from a pyrrole derivative and an indole derivative, and further    comprises a step of neutralization with an organic base after    removal of the temporary protecting group of the 5′-hydroxyl group.-   [35] The production method of any one of [32] to [34], further    comprising the following step (4′):-   (4′) a step of isolating the n′+p′-mer oligonucleotide from the    reaction mixture obtained in step (3′) by an extraction operation    alone.-   [36] The production method of [35], further comprising the following    step (5′):    (5′) a step of removing all the protecting groups of the n′+p′-mer    oligonucleotide obtained in step (4′).

Effect of the Invention

Using the particular oligonucleotide comprising a protected base of thepresent invention, a production method of an oligonucleotide by aphosphoramidite method, which enables efficient purification in a highyield by a liquid-liquid extraction operation, can be provided.

Using the oligonucleotide comprising a protected base, particularly anoligonucleotide comprising a branched chain-protected base, of thepresent invention, liposolubility and solubility in an organic solvent(particularly, non-polar solvent) of an intermediate oligonucleotideobtained in each step of the nucleotide elongation reaction arestrikingly improved to enable isolation and purification by anextraction operation alone, and therefore, a complicated, time-consumingoperation such as solidification isolation and the like is notnecessary, the speed increases, and the efficiency and producibility arestrikingly improved.

Furthermore, since an elongated oligonucleotide can be isolated andpurified by an extraction operation alone, an elongation reaction in thenext cycle can be sequentially performed without taking out theresultant product from the reaction apparatus, whereby anoligonucleotide can be produced continuously in one pot.

It has been clarified that, as another embodiment of the presentinvention, the same kind of solubility improving effect can also beobtained by protecting a nucleotide 3′-hydroxyl group with a protectinggroup having a particular structure of a branched chain-containingaromatic group, and further, a synergistic effect can be obtained by acombined use with an oligonucleotide comprising a protected base.

DESCRIPTION OF EMBODIMENTS

The present invention relates to a novel oligonucleotide comprising aprotected base, wherein the 3′-hydroxyl group is phosphoramidited, the5′-hydroxyl group is protected by a temporary protecting group removableunder acidic conditions, and the nucleic acid base is protected by aprotecting group having a C₅₋₃₀ straight chain or branched chain alkylgroup and/or a C₅₋₃₀ straight chain or branched chain alkenyl group.

In another embodiment, the present invention relates to a productionmethod of an oligonucleotide comprising using an oligonucleotidecomprising a protected base wherein the nucleic acid base is protectedby a group having a C₅₋₃₀ straight chain or branched chain alkyl groupand/or a C₅₋₃₀ straight chain or branched chain alkenyl group, whichpreferably includes the following step (2):

-   (2) a step of condensing a p-mer oligonucleotide comprising a    protected base (p is any integer of one or more) wherein the    3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group is    protected by a temporary protecting group removable under acidic    conditions, and the nucleic acid base is protected by a group having    a C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀    straight chain or branched chain alkenyl group, with an n-mer    oligonucleotide (n is an integer of one or more) wherein the    5′-hydroxyl group is not protected and the 3′-hydroxyl group is    protected, by forming a phosphite triester bond via the 5′-hydroxyl    group thereof to give an n+p-mer oligonucleotide.

In a still another embodiment, the present invention relates to a noveloligonucleotide protected by a branched chain-containing aromatic group,wherein the 3′-hydroxyl group is protected by a particular branchedchain-containing aromatic protecting group, and the 5′-hydroxyl group isprotected by a temporary protecting group removable under acidicconditions.

In a yet another embodiment, the present invention relates to aparticular branched chain-containing aromatic protecting group used forprotecting the nucleotide 3′-hydroxyl group.

In another embodiment, furthermore, the present invention relates to aproduction method of an oligonucleotide, comprising using anoligonucleotide wherein the 3′-hydroxyl group is protected by theaforementioned branched chain-containing aromatic protecting group, andpreferably includes the following step (2′):

-   (2′) a step of condensing a p′-mer oligonucleotide (p′ is any    integer of one or more) wherein the 3′-hydroxyl group is    phosphoramidited, the 5′-hydroxyl group is protected by a temporary    protecting group removable under acidic conditions, and the nucleic    acid base is optionally protected, with an n′-mer oligonucleotide    (n′ is any integer of one or more) wherein the 5′-hydroxyl group is    not protected, and the 3′-hydroxyl group is protected by the    aforementioned branched chain-containing aromatic protecting group,    by forming a phosphite triester bond via the 5′-hydroxyl group    thereof to give an n′+p′-mer oligonucleotide.

Explanations are given below.

1. Explanation of Terms

Unless otherwise specified in the sentences, any technical terms andscientific terms used in the present specification, have the samemeaning as those generally understood by those of ordinary skill in theart the present invention belongs to. Any methods and materials similaror equivalent to those described in the present specification can beused for practicing or testing the present invention, and preferablemethods and materials are described in the following. All publicationsand patents referred to in the specification are hereby incorporated byreference so as to describe and disclose constructed products andmethodology described in, for example, publications usable in relationto the described invention.

In the present specification, the “nucleoside” means a compound whereina nucleic acid base is bonded to the 1′-position of a sugar (e.g.,ribose, 2-deoxyribose, ribose crosslinked between the 2-position and the4-position and the like) by N-glycosidation.

Examples of the ribose wherein the 2-position and the 4-position arecrosslinked include compounds represented by the following formulas.

In the present specification, the “nucleic acid base” is notparticularly limited as long as it can be used for the synthesis ofnucleic acid and includes, for example, a pyrimidine base such ascytosyl group, uracil group, thyminyl group and the like, and a purinebase such as adenyl group, guanyl group and the like.

Moreover, in addition to the above-mentioned groups, a modified nucleicacid base (e.g., a 8-bromoadenyl group, a 8-bromoguanyl group, a5-bromocytosyl group, a 5-iodocytosyl group, a 5-bromouracil group, a5-iodouracil group, a 5-fluorouracil group, a 5-methylcytosyl group, a8-oxoguanyl group, a hypoxanthinyl group etc.), which is a nucleic acidbase substituted by any 1 to 3 substituents (e.g., a halogen atom, analkyl group, an aralkyl group, an alkoxy group, an acyl group, analkoxyalkyl group, a hydroxy group, an amino group, a monoalkylaminogroup, a dialkylamino group, a carboxy group, a cyano group, a nitrogroup etc.) at any position(s), are also encompassed in the “nucleicacid base”.

In the present specification, the “halogen atom” means a fluorine atom,a chlorine atom, a bromine atom or iodine atom.

In the present specification, examples of the “hydrocarbon group”include an aliphatic hydrocarbon group, an aromatic-aliphatichydrocarbon group, a monocyclic saturated hydrocarbon group, an aromatichydrocarbon group and the like. Specific examples thereof include amonovalent group such as an alkyl group, an alkenyl group, an alkynylgroup, a cycloalkyl group, an aryl group, an aralkyl group, an acylgroup and the like, and a divalent group derived therefrom.

In the present specification, examples of the “alkyl (group)” include alinear or branched chain alkyl group having one or more carbon atoms.When the carbon number is not particularly limited, it is preferably aC₁₋₁₀ alkyl group, more preferably a C₁₋₆ alkyl group. When the carbonnumber is not particularly limited, for example, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and thelike are preferable, and methyl and ethyl are particularly preferable.

In the present specification, the “aralkyl (group)” means a C₇₋₂₀aralkyl group, preferably a C₇₋₁₆ aralkyl group (a C₆₋₁₀ aryl-C₁₋₆ alkylgroup).

Preferable specific examples include benzyl, 1-phenylethyl,2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl,1-naphthylpropyl and the like, and benzyl is particularly preferable.

In the present specification, examples of the “alkoxy (group)” includean alkoxy group having one or more carbon atoms. When the carbon numberis not particularly limited, it is preferably a C₁₋₁₀ alkoxy group, morepreferably a C₁₋₆ alkoxy group. When the carbon number is notparticularly limited, methoxy, ethoxy, propoxy, isopropoxy, butoxy,isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like arepreferable, and methoxy and ethoxy are particularly preferable.

In the present specification, examples of the “acyl (group)” include alinear or branched chain C₁₋₆ alkanoyl group, a C₇₋₁₃ aroyl group andthe like. Specific examples thereof include formyl, acetyl, n-propionyl,isopropionyl, n-butyryl, isobutyryl, pivaloyl, valeryl, hexanoyl,benzoyl, naphthoyl, levulinyl and the like, each of which is optionallysubstituted.

In the present specification, examples of the “alkenyl (group)” includea linear or branched chain C₂₋₆ alkenyl group and the like. Preferableexamples thereof include vinyl, 1-propenyl, allyl, isopropenyl, butenyl,isobutenyl and the like. Among them, a C₂-C₄ alkenyl group ispreferable.

In the present specification, examples of the “alkynyl (group)” includea C₂₋₆ alkynyl group and the like. Preferable examples thereof includeethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl,1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl,3-hexynyl, 4-hexynyl, 5-hexynyl and the like. Among them, a C₂-C₄alkynyl group is preferable.

In the present specification, the “cycloalkyl (group)” means a cyclicalkyl group, and examples thereof include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like. Amongthem, a C₃-C₆ cycloalkyl group such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and the like is preferable, and cyclohexyl isparticularly preferable.

In the present specification, the “aryl (group)” means a monocyclicaromatic or polycyclic (fused) hydrocarbon group. Specific examplesthereof include a C₆₋₁₄ aryl group such as phenyl, 1-naphthyl,2-naphthyl, biphenylyl, 2-anthryl and the like, and the like. Amongthem, a C₆₋₄₀ aryl group is more preferably and phenyl is particularlypreferable.

In the present specification, the “organic group having a hydrocarbongroup” means a group having the aforementioned “hydrocarbon group”, andthe moiety other than the “hydrocarbon group” of the “organic grouphaving a hydrocarbon group” can be determined freely. For example, theorganic group optionally has, as a linker, a moiety such as —O—, —S—,—COO—, —OCONH—, —CONH— and the like.

In the present specification, examples of the “alkylene (group)” includea linear or branched chain alkylene group having one or more carbonatoms. When the carbon number is not particularly limited, it ispreferably a C₁₋₁₀ alkylene group, more preferably a C₁₋₆ alkylenegroup. When the carbon number is not particularly limited, for example,methylene, ethylene, propylene, butylene, pentylene, hexylene and thelike are preferable, and methylene and ethylene are particularlypreferable.

2. Oligonucleotide Comprising a Protected Base

The oligonucleotide comprising a protected base to be used foroligonucleotide synthesis in the present invention can be obtained byprotecting a nucleic acid base of a nucleotide wherein the 3′-hydroxylgroup is phosphoramidited and the 5′-hydroxyl group is protected by atemporary protecting group removable under acidic conditions by a grouphaving a C₅₋₃₀ straight chain or branched chain alkyl group and/or aC₅₋₃₀ straight chain or branched chain alkenyl group, and can provide aproduction method of an oligonucleotide suitable for liquid phasesynthesis, since liposolubility and solubility in an organic solvent(particularly, non-polar solvent) of an intermediate oligonucleotide isremarkably improved.

From the aspect of solubility in an organic solvent, the group having aC₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀straight chain or branched chain alkenyl group are/is preferably a C₅₋₃₀branched chain alkyl group and/or a C₅₋₃₀ branched chain alkenyl group.

As a solvent that shows improved solubility of an oligonucleotide by theoligonucleotide comprising a protected base of the presentspecification, a non-polar solvent is preferable.

In the present specification, examples of the “non-polar solvent”include halogenated solvents such as chloroform, dichloromethane,1,2-dichloroethane and the like; aromatic solvents such as benzene,toluene, xylene, mesitylene and the like; ester solvents such as ethylacetate, isopropyl acetate and the like; aliphatic solvents such ashexane, pentane, heptane, octane, nonane, cyclohexane and the like;non-polar ether solvents such as diethyl ether, cyclopentyl methylether, tert-butyl methyl ether and the like can be mentioned. Two ormore kinds of these solvents may be used in a mixture at an appropriateratio. Among them, aromatic solvents, aliphatic solvents and acombination thereof are preferable, benzene, toluene, hexane, pentane,heptane, nonane, cyclohexane, and a combination thereof and the like arepreferable, toluene, heptane, nonane and a combination thereof are morepreferable, and toluene, heptane and a combination thereof areparticularly preferable.

Particularly, in the object method for production of an oligonucleotide,an oligonucleotide comprising a protected base, which is preferable forachieving speeding up, high yield and high efficiency, is, for example,a novel compound represented by the following formula (I) (hereinaftersometimes to be referred to as the compound (I) of the presentinvention).

When q is 0, the oligonucleotide comprising a protected base, which isrepresented by the formula (I), is understood as “nucleoside comprisinga protected base”.

Formula (I):

-   wherein q is any integer of not less than 0;-   Base² in the number of q+1 are each independently a nucleic acid    base protected by a group having a C₅₋₃₀ straight chain or branched    chain alkyl group and/or a C₅₋₃₀ straight chain or branched chain    alkenyl group;-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    halogen atom or an organic group crosslinked with the 4-position    carbon atom;-   X′ in the number of q are each independently a hydrogen atom, an    optionally protected hydroxyl group, a halogen atom or an organic    group crosslinked with the 4-position carbon atom;-   P² in the number of q+1 are each independently a protecting group    removable under basic conditions;-   R³⁴ in the number of q are each independently an oxygen atom or a    sulfur atom; and-   R_(e) and R_(f) are each independently a C₁₋₆ alkyl group, or a 5-    or 6-membered saturated cyclic amino group formed together with the    adjacent nitrogen atom.

q is any integer of 0 or more, preferably 0. While the upper limit of qis not particularly set, it is preferably 49 or less, more preferably 29or less, further preferably 19 or less, still more preferably 4 or less,still further preferably 2 or less and particularly preferably 1.

The groups X and X′ in the number of q at the 2-position of riboseresidues constituting the oligonucleotide comprising a protected base,which is the compound (I) of the present invention, are eachindependently a hydrogen atom, an optionally protected hydroxyl group, ahalogen atom or an organic group crosslinked with the 4-position carbonatom.

The protecting group of the “optionally protected hydroxyl group” is notparticularly limited and, for example, any protecting groups describedin Greene's PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4th ed., JOHNWILLY&SONS (2006) and the like can be mentioned. Specific examplesthereof include a methyl group, a benzyl group, a p-methoxybenzyl group,a tert-butyl group, a methoxymethyl group, a methoxyethyl group, a2-tetrahydropyranyl group, an ethoxyethyl group, a cyanoethyl group, acyanoethoxymethyl group, a phenylcarbamoyl group, a1,1-dioxothiomorpholine-4-thiocarbamoyl group, an acetyl group, apivaloyl group, a benzoyl group, a trimethylsilyl group, a triethylsilylgroup, a triisopropylsilyl group, a tert-butyldimethylsilyl group, a[(triisopropylsilyl)oxy]methyl (Tom) group, an1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group and the like.Among these, a triethylsilyl group, a triisopropylsilyl group and atert-butyldimethylsilyl group are preferable. From the aspects ofeconomic efficiency and easy availability, a tert-butyldimethylsilylgroup is particularly preferable.

As the halogen atom for X or X′, a fluorine atom, a chlorine atom andthe like are preferable, and a fluorine atom is more preferable.

While the “organic group crosslinked with the 4-position carbon atom”for X or X′ is not particularly limited as long as the 2-position andthe 4-position of the nucleoside is crosslinked, for example, a C₂₋₇alkylene group can be mentioned. The alkylene group may be interruptedat one or more moieties (preferably 1 or 2 moieties) by a linkerselected from, for example, —O—, —NR³⁷— (R³⁷ is a hydrogen atom or aC₁₋₆ alkyl group), —S—, —CO—, —COO—, —COO—, NR³⁸— (R³⁸ is a hydrogenatom or a C₁₋₆ alkyl group), —CONR³⁹— (R³⁹ is a hydrogen atom or a C₁₋₆alkyl group) and the like.

Preferable examples of the “organic group crosslinked with the4-position carbon atom” include —ORi (Ri is a C₁₋₆ alkylene groupcrosslinked with the 4-position), —O—NR³⁷-Rj (Rj is a C₁₋₆ alkylenegroup crosslinked with the 4-position, and R³⁷ is as defined above),—O—Rk-O-Rl (Rk is a C₁₋₆ alkylene group, and Rl is a C₁₋₆ alkylene groupcrosslinked with the 4-position) and the like. The C₁₋₆ alkylene groupsfor Ri, Rj, Rk and Rl are preferably each independently a methylenegroup or an ethylene group.

As the “organic group crosslinked with the 4-position carbon atom”,—O—CH₂—, —O—CH₂—CH₂—, —O—NR³⁷—CH₂— (R³⁷ is as defined above),—O—CH₂—O—CH₂— and the like are preferable, and —O—CH₂—, —O—CH₂—CH₂—,—O—NH—CH₂—, —O—NMe-CH₂—, —O—CH₂—O—CH₂— (in all of which the left sidebinds to the 2-position and the right side binds to the 4-position) andthe like are more preferable.

The temporary protecting group P¹ that can be used as the5′-hydroxyl-protecting group of the compound (I) of the presentinvention is not particularly limited as long as it can be deprotectedunder acidic conditions and can be used as a hydroxyl-protecting group.Examples thereof include a trityl group, 9-(9-phenyl)xanthenyl group, a9-phenylthioxanthenyl group, di(C₁₋₆ alkoxy)trityl groups such as a1,1-bis(4-methoxyphenyl)-1-phenylmethyl group and the like,monomethoxytrityl group such as mono(C₁₋₁₈ alkoxy)trityl groups such asa 1-(4-methoxyphenyl)-1,1-diphenylmethyl group and the like. Amongthese, a monomethoxytrityl group and a dimethoxytrityl group arepreferable, and a dimethoxytrityl group is more preferable, in view ofeasiness of deprotection and easy availability.

The 5- or 6-membered saturated cyclic amino group formed by R_(e) andR_(f) together with the adjacent nitrogen atom may have, as aring-constituting atom besides nitrogen atom, one of oxygen atom orsulfur atom. For example, a pyrrolidinyl group, a piperidinyl group, amorpholinyl group, a thiomorpholinyl group, an N-methylpyrazinyl groupand the like can be mentioned.

As R_(e) and R_(f), an isopropyl group is preferable.

The protecting group P² in the number of q+1 that can be used as aprotecting group of phosphoramidite or a protecting group of anucleotide phosphate group of the compound (I) of the present inventionis not particularly limited as long as it can be deprotected under baseconditions and used as a hydroxyl-protecting group. A group representedby —CH₂CH₂WG (WG is an electron-withdrawing group) is preferable, and acyano group is preferable as WG. R³⁴ in the number of q is eachindependently an oxygen atom or a sulfur atom, preferably an oxygenatom.

While the “group having a C₅₋₃₀ straight chain or branched chain alkylgroup and/or a C₅₋₃₀ straight chain or branched chain alkenyl group” ofthe “nucleic acid base protected by a group having a C₅₋₃₀ straightchain or branched chain alkyl group and/or a C₅₋₃₀ straight chain orbranched chain alkenyl group” for Base² in the number of q+1 is notparticularly limited as long as it is a protecting group having one ormore “C₅₋₃₀ straight chain or branched chain alkyl group and/or C₅₋₃₀straight chain or branched chain alkenyl group” in the molecularstructure thereof, a group represented by the following formula (k),(l), (m) or (s) is preferable.

A group represented by the formula (k):

-   wherein * indicates the bonding position to a nucleic acid base;-   R²⁷ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (l):

-   wherein * indicates the bonding position to a nucleic acid base;-   Q₁ is —O—, —S— or —NR³⁰— wherein R³⁰ is a hydrogen atom or a C₁₋₂₂    alkyl group;-   R_(c) and R_(d) are each independently a hydrogen atom or a C₁₋₂₂    alkyl group;-   R²⁸ is a C₅₋₃₀ straight chain or branched chain alkyl group or a    C₅₋₃₀ straight chain or branched chain alkenyl group,-   a group represented by the formula (m):

-   wherein * indicates the bonding position to a nucleic acid base;-   l is an integer of 1-5;-   Q₂ in the number of l are each independently a single bond, or —O—,    —S—, —OC(═O)—, —C(═O)O—, —O—CH₂—, —NH—, —NHC(═O)—, —(═O)NHC—,    —NH—CH₂— or —CH₂—;-   R²⁹ in the number of l are each independently a C₅₋₃₀ straight chain    or branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group;-   ring C is a benzene ring or a cyclohexane ring each optionally    having, in addition to Q₂R²⁹ in the number of l and *C═O, a    substituent selected from the group consisting of a halogen atom, a    C₁₋₆ alkyl group optionally substituted by one or more halogen    atoms, and a C₁₋₆ alkoxy group optionally substituted by one or more    halogen atoms, or-   a group represented by the formula (s):

-   wherein * indicates the position at which an imino bond is formed    with an amino group of a nucleic acid base; and-   R³⁵ and R³⁶ are each independently a C₅₋₃₀ straight chain or    branched chain alkyl group or a C₅₋₃₀ straight chain or branched    chain alkenyl group.

In the formula (l), Q₁ is preferably —O—, and R_(c) and R_(d) are eachpreferably a hydrogen atom.

In the formula (m), l is preferably 1, Q₂ is preferably —O—, and ring Cis preferably a benzene ring.

The lower limit of the carbon atoms of the “C₅₋₃₀ straight chain orbranched chain alkyl group” and “C₅₋₃₀ straight chain or branched chainalkenyl group” of the “C₅₋₃₀ straight chain or branched chain alkylgroup or a C₅₋₃₀ straight chain or branched chain alkenyl group” forR²⁷, R²⁸, R²⁹ in the number of l, R³⁵ and R³⁶ in the formulas (k), (l),(m) and (s), respectively, is 5 or more, preferably 16 or more, morepreferably 18 or more. The upper limit of the carbon number is 30 orless, preferably or less, more preferably 20 or less.

While the “C₅₋₃₀ straight chain or branched chain alkyl group or C₅₋₃₀straight chain or branched chain alkenyl group” for R²⁷, R²⁸, R²⁹ in thenumber of l, R³⁵ or R³⁶ is not particularly limited, a branched chainalkyl group represented by the following formula (n) or (e′) ispreferable.

The carbon numbers, the number of repeat units (n₁₉, n₂₀ or n₂₁) and thelike in the definition of each symbol in the formulas (n) and (e′) areshown for convenience, and can be appropriately changed within theabove-mentioned definition range to achieve the total carbon number of 5or more (preferably 16 or more, more preferably 18 or more) and 30 orless (preferably 25 or less, more preferably 20 or less). In thefollowing, the formulas (n) and (e′) are successively explained.

The formula (n) is as follows.

A branched chain alkyl group represented by

-   wherein * shows a bonding position;-   n₁₉ is an integer of 2 to 6;-   R³⁰ and R³¹ in the number of n₁₉ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₆ in the number of n₁₉ are each independently a single bond or a    C₁₋₄ alkylene group; and-   R³² is a hydrogen atom or a C₁₋₄ alkyl group; and-   R³³ is a C₁₋₄ alkyl group,-   provided that R³⁰ and R³¹ are not hydrogen atoms at the same time,    and when n₁₉ is 2, R₃₂ is a C₁₋₄ alkyl group.

In the group of the formula (n), a group wherein R³⁰ and R³¹ in thenumber of n₁₉ are each independently a hydrogen atom, a methyl group oran ethyl group;

-   X₆ in the number of n₁₉ are each independently a single bond, a    methylene group or an ethylene group;-   R³² is a hydrogen atom, a methyl group or an ethyl group; and-   R³³ is a methyl group or an ethyl group is preferable, provided that    R³⁰ and R³¹ are not hydrogen atoms at the same time, and when n₁₉ is    2, R₃₂ is a methyl group or an ethyl group.

Examples of more preferable group of the formula (n) include a2,6,10,14-tetramethylpentadecyl group, a 2,6,10-trimethylundecyl group,a 2,6-dimethylheptyl group and the like.

A branched chain alkyl group represented by the formula (e′):

-   wherein * shows a bonding position;-   n₂₀ is an integer of 1 to 5;-   n₂₁ is an integer of 1 to 5;-   R²⁰ and R²¹ in the number of n₂₀ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₃ in the number of n₂₀ are each independently a single bond or a    C₁₋₄ alkylene group;-   R²² and R²³ in the number of n₂₁ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₅ in the number of n₂₁ are each independently a single bond or a    C₁₋₄ alkylene group;-   X₄ is a single bond or a C₁₋₄ alkylene group; and-   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵ and R²⁶ are each independently a hydrogen    atom or a C₁₋₄ alkyl group,-   provided that R²⁰ and R²¹ and/or R²² and R²³ are not hydrogen atoms    at the same time, and when n₂₀+n₂₁ is 2, two or more of R¹⁷, R¹⁸ and    R¹⁹ are each independently a C₁₋₄ alkyl group, or two or more of    R²⁴, R²⁵ and R²⁶ are each independently a C₁₋₄ alkyl group.

A group of the formula (e′), wherein

-   n₂₀ is an integer of 1 to 5;-   n₂₁ is an integer of 1 to 5;-   R²⁰ and R²¹ in the number of n₂₀ are each independently a hydrogen    atom, a methyl group or an ethyl group;-   X₃ in the number of n₂₀ are each independently a single bond, a    methylene group or an ethylene group;-   R²² and R²³ in the number of n₂₁ are each independently a hydrogen    atom, a methyl group or an ethyl group;-   X₅ in the number of n₂₁ are each independently a single bond, a    methylene group or an ethylene group;-   X₄ is a single bond, a methylene group or an ethylene group;-   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵ and R²⁶ are each independently a hydrogen    atom or a C₁₋₄ alkyl group is preferable, provided that R²⁰ and R²¹    and/or R²² and R²³ are not hydrogen atoms at the same time, and when    n₂₀+n₂₁ is 2, two or more of R¹⁷, R¹⁸ and R¹⁹ are each independently    a C₁₋₄ alkyl group, or two or more of R²⁴, R²⁵ and R²⁶ are each    independently a C₁₋₄ alkyl group.

A group of the formula (e′), wherein

-   n₂₀ is an integer of 1 to 5;-   n₂₁ is an integer of 1 to 5;-   R²⁰ and R²¹ in the number of n₂₀ are each independently a hydrogen    atom or a methyl group;-   X₃ in the number of n₂₀ are each independently a single bond or a    methylene group;-   R²² and R²³ in the number of n₂₁ are each independently a hydrogen    atom or a methyl group;-   X₅ in the number of n₂₁ are each independently a single bond or a    methylene group;-   X₄ is a single bond or a methylene group; and-   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵ and R²⁶ are methyl groups, provided that R²⁰    and R²¹ and/or R²² and R²³ are not hydrogen atoms at the same time,    is particularly preferable.

More preferable group of the formula (e′) includes a2,2,4,8,10,10-hexamethyl-5-undecyl group and the like.

Other preferable examples of the “C₅₋₃₀ straight chain or branched chainalkyl group or C₅₋₃₀ straight chain or branched chain alkenyl group” forR²⁷, R²⁸, R²⁹ in the number of l, R³⁵ or R³⁶ include a branched chainalkyl group and a branched chain alkenyl group selected from the groupconsisting of a 2,6,10,14-tetramethylpentadecyl group, a2,6,10-trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecylgroup, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a2,6-dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a2,6-dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a12-methyltridecyl group, an 11-methyltridecyl group, an 11-methyldodecylgroup, a 10-methylundecyl group, an 8-heptadecyl group, a 7-pentadecylgroup, a 7-methyloctyl group, a 3-methyloctyl group, a 3,7-dimethyloctylgroup, a 3-methylheptyl group, a 3-ethylheptyl group, a 5-undecyl group,a 2-heptyl group, a 2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptylgroup, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methyl-pentylgroup, and a 2,4,4-trimethylpentyl group; and a straight chain alkylgroup selected from the group consisting of a tetradecyl group, atridecyl group, a dodecyl group, an undecyl group, a decyl group, anonyl group, an octyl group, a heptyl group, a hexyl group, and a pentylgroup.

Examples of the nucleic acid base protected by a group having a C₅₋₃₀straight chain or branched chain alkyl group and/or a C₅₋₃₀ straightchain or branched chain alkenyl group include the following formulas(A₁)-(A₁₂)

wherein each symbol is as defined above.

A method of introducing a group having a C₅₋₃₀ straight chain orbranched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group into a nucleic acid base can be performed accordingto a known method described in Greene's PROTECTIVE GROUPS IN ORGANICSYNTHESIS, 4th Edition, published by JOHN WILLY & SONS (2006), ORGANICLETTERS, 2005, Vol. 7, No. 24, 5389-5392, JOURNAL OF AMERICAN CHEMICALSOCIETY, 1982, Vol. 104, 1316-1319 and the like and using, as aprotecting reagent, an activated derivative of the protecting group.

Examples of the activated derivative of the group having a C₅₋₃₀straight chain or branched chain alkyl group and/or a C₅₋₃₀ straightchain or branched chain alkenyl group include a compound wherein ahalogen atom (e.g., chlorine atom, bromine atom etc.) is bonded to * ina group represented by the above-mentioned formula (k), (l) or (m); asymmetric acid anhydride wherein two groups represented by theabove-mentioned formula (k) or (m) are bonded to an oxygen atom at *; amixed acid anhydride wherein a group represented by the above-mentionedformula (k) or (m) is bonded to other acyl group (e.g., isobutyrylgroup) at *, a compound represented by (MeO)₂CH—NR³⁵R³⁶ and the like.

The activated derivative of the protecting group is available as acommercially available product, or can be produced by a method known perse or a method analogous thereto.

A preferable embodiment of the compound represented by the formula (I)of the present invention is a compound of the formula (I), wherein

-   q is 0;-   Base² is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected by a group having a C₅₋₃₀ straight chain or branched chain    alkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenyl    group;-   P¹ is a di (C₁₋₆ alkoxy)trityl group, or a mono (C₁₋₆ alkoxy)trityl    group;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    fluorine atom, —ORi (Ri is as defined above), —O—NR³⁷—Rj (Rj and R³⁷    are as defined above, or —O—Rk-O—Rl (Rk and Rl is as defined above);-   R_(e) and R_(f) are each independently a C₁₋₆ alkyl group; and-   P² is a group represented by —CH₂CH₂WG (WG is an    electron-withdrawing group).

Another preferable embodiment of the compound represented by the formula(I) of the present invention is a compound of the formula (I), wherein

-   q is 0;-   Base² is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected by a group having a C₅₋₃₀ straight chain or branched chain    alkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenyl    group;-   P¹ is a dimethoxytrityl group or a monomethoxytrityl group;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    fluorine atom, —O—CH₂—, —O—CH₂—CH₂—, or —O—NR³⁷—CH₂— (R³⁷ is as    defined above) or —O—CH₂—O—CH₂— (in all of which the left side binds    to the 2-position and the right side binds to the 4-position);-   R_(e) and R_(f) are each an isopropyl group; and-   P² is a group represented by —CH₂CH₂WG (WG is an    electron-withdrawing group).

A still another preferable embodiment of the compound represented by theformula (I) of the present invention is a compound of the formula (I),wherein

-   q is 0;-   Base² is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected by a group having a C₅₋₃₀ straight chain or branched chain    alkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenyl    group;-   P¹ is a dimethoxytrityl group;-   X is a hydrogen atom, a methoxy group, a tert-butyldimethylsilyloxy    group, a fluorine atom, —O—CH₂—, —O—CH₂—CH₂—, —O—NH—CH₂—,    —O—NMe-CH₂— or —O—CH₂—O—CH₂— (in all of which the left side binds to    the 2-position and the right side binds to the 4-position);-   R_(e) and R_(f) are each an isopropyl group; and-   P² is a group represented by —CH₂CH₂CN.    3. Production Method of Oligonucleotide Comprising Protected Base

Of the oligonucleotides comprising a protected base represented by thefollowing formula (I) of the present invention, an oligonucleosidecomprising a protected base represented by the formula (I′), wherein qis 0, can be produced according to a known method (M. H. Caruthers etal., Method in Enzymology 1987, 154, 287-313; S. L. Beaucage and M. H.Caruthers, Tetrahedron Letters 1981, 22, 1859-1862) comprising reactinga nucleoside represented by the formula (Ia), wherein the 5′-hydroxylgroup is protected by a temporary protecting group P¹ and the nucleicacid base is protected by a group having a C₅₋₃₀ straight chain orbranched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group, with a phosphoramiditing reagent represented by thefollowing formula (o) or (p)

-   wherein Hal is a halogen atom, and other symbols are as defined    above.

A compound represented by the formula (Ia) to be used as a startingmaterial can be produced from a corresponding nucleoside wherein thenucleic acid base is not protected, by protecting the nucleic acid basewith a “group having a C₅₋₃₀ straight chain or branched chain alkylgroup and/or a C₅₋₃₀ straight chain or branched chain alkenyl group”according to the above-mentioned “method for introducing a group havinga C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀straight chain or branched chain alkenyl group into a nucleic acidbase”. The corresponding nucleoside wherein the nucleic acid base is notprotected is available as a commercially available product, or can beproduced according to a method known per se or a method analogousthereto.

The nucleoside comprising a protected base represented by the formula(I′) of the present invention can also be produced by a known method(ORGANIC LETTERS, 2005, Vol. 7, No. 24, 5389-5392) comprising reacting anucleoside represented by the formula (Ib), wherein the 3′-hydroxylgroup is phosphoramidited, the 5′-hydroxyl group is protected by atemporary protecting group P¹, and the nucleic acid base is notprotected, with a protecting reagent having a C₅₋₃₀ straight chain orbranched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group.

-   wherein Base′ is an unprotected nucleic acid base, and other symbols    are as defined above.

A compound represented by the formula (Ib) to be used as a startingmaterial can be produced by deprotecting a corresponding nucleosidewherein the nucleic acid base is protected by a protecting groupconventionally used for nucleic acid synthesis (e.g., acetyl group,phenoxyacetyl group, p-isopropylphenoxyacetyl group, benzoyl group,isobutyryl group etc.) according to a known method described in ORGANICLETTERS, 2005, Vol. 7, No. 24, 5389-5392 and the like. The correspondingnucleoside wherein the nucleic acid base is protected by a protectinggroup conventionally used for nucleic acid synthesis is available as acommercially available product, or can be produced according to a methodknown per se or a method analogous thereto.

An oligonucleotide comprising a protected base represented by of theformula (I) wherein q is 1 or more can be produced by appropriatelyapplying, for example, the method described in Aust. J. Chem. 2010, 63,227-235 and the production method of oligonucleoside described in thepresent specification to produce a corresponding oligonucleotidecomprising a protected base wherein the 3′-hydroxyl group is protected,removing the 3′-hydroxyl-protecting group, and reacting the resultingcompound with a phosphoramiditing reagent represented by the formula (o)or (p) according to a known method (M. H. Caruthers et al., Method inEnzymology 1987, 154, 287-313; S. L. Beaucage and M. H. Caruthers,Tetrahedron Letters 1981, 22, 1859-1862.).

-   wherein P⁴ is a nucleotide 3′-hydroxyl-protecting group, and other    symbols are as defined above.

As the protecting group of nucleotide 3′-hydroxyl group for P⁴, aprotecting group capable of deprotection under the conditions underwhich the 5′-hydroxyl-protecting group P¹, the protecting group when thenucleic acid base has a protecting group and a phosphate-protectinggroup P² are not deprotected is used. For example, a protecting groupcapable of deprotection with hydrazine can be mentioned. Preferableexamples of the protecting group capable of deprotection with hydrazineinclude a levulyl group and the like (see Aust. J. Chem. 2010, 63,227-235).

4. Production Method of Oligonucleotide

Next, the production method of oligonucleotide relating to the presentinvention (hereinafter to be also referred to as “the production methodof the present invention”) is explained.

The production method of the present invention characteristicallycomprises using the aforementioned oligonucleotide comprising aprotected base. To be specific, a production method of an n+p-meroligonucleotide from an n-mer oligonucleotide is explained. For example,when n=1, an n-mer oligonucleotide is to be understood as “nucleoside”,when p=1, a p-mer oligonucleotide comprising a protected base is to beunderstood as “nucleoside comprising a protected base”, and an n+p-meroligonucleotide is to be understood as “dinucleoside”.

The production method of the present invention preferably includes thefollowing step (2):

-   (2) a step of condensing a p-mer oligonucleotide comprising a    protected base (p is any integer of one or more) wherein the    3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group is    protected by a temporary protecting group removable under acidic    conditions, and the nucleic acid base is protected by a group having    a C₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀    straight chain or branched chain alkenyl group, with an n-mer    oligonucleotide (n is an integer of one or more) wherein the    5′-hydroxyl group is not protected and the 3′-hydroxyl group is    protected, by forming a phosphite triester bond via the 5′-hydroxyl    group thereof to give an n+p-mer oligonucleotide.

The production method of the present invention preferably furtherincludes the following step (3), by which the phosphite triester bond ofthe n+p-mer oligonucleotide obtained in step (2) is converted to aphosphate triester bond or thiophosphate triester bond.

-   (3) a step of converting the phosphite triester bond of the n+p-mer    oligonucleotide obtained in the condensation step to a phosphate    triester bond or a thiophosphate triester bond by adding an    oxidizing agent or a sulfurizing agent to the reaction mixture    obtained in the condensation step (2).

The production method of the present invention preferably furtherincludes the following step (1), whereby an n-mer oligonucleotidewherein the 5′-hydroxyl group is not protected and the 3′-hydroxyl groupis protected, which is used in step (2), is prepared:

-   (1) a step of removing the temporary protecting group removable    under acidic conditions of the 5′-hydroxyl group by reacting, in a    non-polar solvent prior to the condensation step (2), an n-mer    oligonucleotide wherein the 3′-hydroxyl group is protected, and the    5′-hydroxyl group is protected by a temporary protecting group, with    an acid.

Step (1) is preferably performed in the presence of at least one kind ofcation scavenger selected from a pyrrole derivative and an indolederivative, and further includes a step of removing the temporaryprotecting group of the 5′-hydroxyl group and neutralizing the compoundwith an organic base. This enables continuous performance of steps (1),(2) and (3) in a solution, and an oligonucleotide wherein nucleoside inthe number of p has elongated can be isolated and purified by anextraction operation alone.

Furthermore, by including the following step (4), an n+p-meroligonucleotide is purified by removing an excess starting material andby-product conveniently and effectively, without the need forcomplicated solidification-isolation, and can be led to the next stepwithout taking out the resultant product from the reaction vessel:

-   (4) a step of isolating the n+p-mer oligonucleotide from the    reaction mixture obtained in step (3) by an extraction operation    alone.

When the amount of the by-product can be controlled by the management ofequivalent amounts of the starting materials and the control of thereaction, it is preferable to repeat step (1) to step (3) as a basicunit, and include step (4).

Moreover, since occurrence of by-product can be strictly managed andcontrolled and highly pure oligonucleotide can be obtained, it ispreferable to repeat step (1) to step (4) as a basic unit.

By repeating such cycle in the liquid phase method, the finaloligonucleotide can be produced in one-pot, without changing thereaction vessel.

In the production method of the present invention, an oligonucleotidecan be isolated and produced by further including step (5):

-   (5) a step of removing all the protecting groups of the n+p-mer    oligonucleotide obtained in step (4).

n is an integer of one or more. While the upper limit thereof is notparticularly limited, it is generally 100 or less, preferably 75 orless, more preferably 50 or less, and still more preferably 30 or less

p is an integer of one or more, preferably 1. While the upper limitthereof is not particularly limited, it is preferably 50 or less, morepreferably 30 or less, more preferably 20 or less, still more preferably5 or less, and particularly preferably 3 or less.

4-1. Explanation of “n-mer oligonucleotide”

First of all, the n-mer oligonucleotide to be used as a startingmaterial of steps (1) and (2) is explained.

The n-mer oligonucleotide to be used in step (1) is, for example, ann-mer oligonucleotide represented the following formula (i) wherein P¹is a temporary protecting group removable under acidic conditions, the3′-position hydroxyl group is protected and the 5′-position hydroxylgroup is protected by a temporary protecting group removable underacidic conditions. The n-mer oligonucleotide to be used in step (2)shows, for example, an n-mer oligonucleotide represented by thefollowing formula (II) wherein the 5′-position hydroxyl group is notprotected, and the 3′-position hydroxyl group is protected.

-   wherein m is any integer of not less than 0, Base in the number of    m+l are each independently an optionally protected nucleic acid    base, R³⁴ in the number of m are each independently an oxygen atom    or a sulfur atom, P² in the number of m are each independently a    protecting group removable under basic conditions, P³ is a    nucleotide 3′-hydroxyl-protecting group, X′ in the number of m+1 are    each independently as defined for X, and other symbols are as    defined above.

While the upper limit of m is not particularly limited, it is generally99 or less, preferably 74 or less, more preferably 49 or less, stillmore preferably 29 or less.

The “protecting group removable under basic conditions” for P² is asdefined for P² in the formula (I).

Each symbol in the formulas (i) and (ii) is explained below.

4-2. Explanation of “Nucleotide 3′-Hydroxyl-Protecting Group”

The “nucleotide 3′-hydroxyl-protecting group” for P³ in the formulas (i)and (ii) is not particularly limited as long as it is a group stableunder acidic conditions capable of removing the 5′-hydroxyl-protectinggroup, and can dissolve an n-mer oligonucleotide in a non-polar reactionsolvent so that the reaction will proceed in steps (1) and (2). It ispreferably a group represented by the following formula (III′)-L-Y—Z′  (III′)wherein

-   L is a group represented by the formula (a1):

-   wherein * shows the bonding position to Y; ** indicates the bonding    position to a 3′-hydroxy group of the nucleotide;-   L₁ is an optionally substituted divalent C₁₋₂₂ hydrocarbon group;    and-   L₂ is a single bond, or a group represented by    **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows the bonding position to    L₁, *** shows the bonding position to C═O, R¹ is an optionally    substituted C₁₋₂₂ alkylene group, and R² and R³ are each    independently a hydrogen atom or an optionally substituted C₁₋₂₂    alkyl group, or-   R² and R³ are optionally joined to form an optionally substituted    C₁₋₂₂ alkylene bond,-   Y is an oxygen atom or NR wherein R is a hydrogen atom, an alkyl    group or an aralkyl group, and-   Z′ is an organic group having a hydrocarbon group.

A preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group or CH₂—O-1,4-phenylene-O—CH₂; and-   L₂ is a single bond, or a group represented by    **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows the bonding position to    L₁, *** shows the bonding position to C═O, R¹ is a C₁₋₆ alkylene    group, and R² and R³ are each independently a hydrogen atom or an    optionally substituted C₁₋₆ alkyl group, or R² and R³ are optionally    joined to form an optionally substituted C₁₋₆ alkylene bond.

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group; and-   L₂ is a single bond.

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group; and-   the moiety N(R²)—R¹—N(R³) for L₂ is a piperazinylene group.

Another preferable embodiment of the linker L represented by theabove-mentioned formula (a1) is a group wherein, in the formula (a1),

-   L₁ is an ethylene group; and-   L₂ is a group represented by **C(═O)N(R²)—R¹—N(R³)*** wherein **    shows the bonding position to L₁, *** shows the bonding position to    C═O, R¹ is a pentylene group or a hexylene group, and R² and R³ are    each independently a hydrogen atom or a methyl group.

A particularly preferable example of the above-mentioned linker L is asuccinyl group since it is economical and easily available.

Y in the above-mentioned formula (III′) is an oxygen atom, or NR whereinR is a hydrogen atom, an alkyl group or an aralkyl group.

In the present specification, the “alkyl group” for R is a C₁₋₃₀ alkylgroup, preferably a C₁₋₁₀ alkyl group, more preferably a C₁₋₆ alkylgroup. Specific preferable examples thereof include methyl, ethyl,propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl and the like,and methyl and ethyl are particularly preferable.

In the present specification, the “aralkyl group” for R is a C₇₋₃₀aralkyl group, preferably a C₇₋₂₀ aralkyl group, more preferably a C₇₋₁₆aralkyl group (a C₆₋₁₀ aryl-C₁₋₆ alkyl group). Specific preferableexamples thereon include benzyl, 1-phenylethyl, 2-phenylethyl,1-phenylpropyl, α-naphthylmethyl, 1-(α-naphthyl)ethyl,2-(α-naphthyl)ethyl, 1-(α-naphthyl)propyl, p-naphthylmethyl,1-(β-naphthyl)ethyl, 2-(β-naphthyl)ethyl, 1-(β-naphthyl)propyl and thelike, and benzyl is particularly preferable.

R is preferably a hydrogen atom, a C₁₋₆ alkyl group or a C₇₋₁₆ aralkylgroup, more preferably a hydrogen atom, methyl, ethyl or benzyl,particularly preferably a hydrogen atom.

Y is preferably an oxygen atom or NH.

Examples of the “organic group having a hydrocarbon group” for Z′include a C₁₋₆ alkyl group such as methyl, ethyl, tert-butyl and thelike, benzyl, p-nitrobenzyl, p-methoxybenzyl, diphenylmethyl, allyl,1,1-dimethyl-2-phenyl-ethyl, 2,4-dimethoxybenzyl,bis(4-methoxyphenyl)methyl and the like. In addition, as the organicgroup having a hydrocarbon group, a group having a branched chain ispreferable. When a group having a branched chain is used, liposolubilityand solubility in a solvent (particularly, non-polar solvent) of ann-mer oligonucleotide and an n+p-mer oligonucleotide can be improved,step (1) can be performed smoothly, and an n+p-mer oligonucleotide canbe easily transferred into a non-polar solvent in theextraction-isolation step of the below-mentioned step (4).

Preferable examples of the “group having a branched chain” for Z′include a group represented by the formula (a2):

-   wherein * shows the bonding position to Y;-   R⁴ is a hydrogen atom, or when R_(b) is a group represented by the    following formula (a3), then R⁴ shows, in combination with R⁶, a    single bond or —O— to optionally form a fluorenyl group or a    xanthenyl group together with ring B;-   Q in the number of k are each independently a single bond, —O—, —S—,    —OC(═O)—, —NHC(═O)— or —NH—;-   R⁵ in the number of k are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   k is an integer of 1 to 4;-   ring A optionally further has, in addition to R⁴, QR⁵ in the number    of k and *C(R_(a))(R_(b)), a substituent selected from the group    consisting of a halogen atom, a C₁₋₆ alkyl group optionally    substituted by one or more halogen atoms, and a C₁₋₆ alkoxy group    optionally substituted by one or more halogen atoms;-   R_(a) is a hydrogen atom; and-   R_(b) is a hydrogen atom, or a group represented by the formula    (a3):

-   wherein * indicates the bonding position;-   j is an integer of 0 to 4;-   Q in the number of j are each independently as defined above;-   R⁷ in the number of j are each independently an organic group having    at least one aliphatic hydrocarbon group having one or more branched    chains and the total carbon number of not less than 14 and not more    than 300;-   R⁶ is a hydrogen atom, or shows, in combination with R⁴, a single    bond or —O— to form a fluorenyl group or a xanthenyl group together    with ring A; and-   ring B optionally further has, in addition to QR⁷ in the number of j    and R⁶, a substituent selected from the group consisting of a    halogen atom, a C₁₋₆ alkyl group optionally substituted by one or    more halogen atoms, and a C₁₋₆ alkoxy group optionally substituted    by one or more halogen atoms.

That is, a preferable one embodiment of the 3′-hydroxyl-protecting groupin the n-mer oligonucleotide in the present invention is represented bythe following formula (III):-L-Y—Z  (III)wherein

-   L and Y are as defined above, and-   Z is the formula (a2):

wherein each symbol is as defined above.

When the nucleotide 3′-hydroxyl-protecting group is -L-Y—Z′ and Z′ is a“group having branched chain”, preferably a group represented by theformula (a2), then a solvent, wherein solubility of the nucleoside oroligonucleotide is improved, is preferably a non-polar solvent.

Preferable embodiments of L and Y in the above-mentioned formula (III)are similar to those of the above-mentioned formula (III′).

The preferable embodiment for Z′ in the above-mentioned formula (III′),that is, a group represented by the formula (a2) for Z in theabove-mentioned formula (III) is a particular benzyl group (in theformula (a2), both R_(a) and R_(b) are hydrogen atoms, and R⁴ is ahydrogen atom); a particular diphenylmethyl group (in the formula (a2),R_(a) is a hydrogen atom, R⁴ is a hydrogen atom, k is 1 to 3, and R_(b)is a group represented by the formula (a3) wherein R⁶ is a hydrogenatom, and j is 0 or 1); a particular fluorenyl group (in the formula(a2), R_(a) is a hydrogen atom, k is 1, R_(b) is a group represented bythe formula (a3) wherein j is 0, and R⁶ shows, together with R⁴, asingle bond to form a fluorine ring together with ring A); a particularxanthenyl group (in the formula (a2), R_(a) is a hydrogen atom, k is 1,R_(b) is a group represented by the formula (a3) wherein j is 0, and R⁶shows —O— together with R⁴ to form a xanthine ring together with ringA).

In the formula (III), the “organic group having at least one aliphatichydrocarbon group having one or more branched chains and a total carbonnumber of not less than 14 and not more than 300” for R⁵ and R⁷ is anorganic group having at least one aliphatic hydrocarbon group having oneor more branched chains in a molecular structure thereof, and a totalcarbon number of not less than 14 and not more than 300.

The “branched chain” of the “aliphatic hydrocarbon group having one ormore branched chains” is a straight or branched saturated aliphatichydrocarbon group. Preferred is a C₁₋₆ alkyl group, more preferred is aC₁₋₄ alkyl group, and still more preferred is a methyl group or an ethylgroup. In addition, the “branched chain” is optionally substituted byone or more halogen atoms.

The “aliphatic hydrocarbon group” of the “aliphatic hydrocarbon grouphaving one or more branched chains” is a straight saturated orunsaturated aliphatic hydrocarbon group, a C₂-C₃₀₀ alkyl group(preferably, a C₃-C₁₀₀ alkyl group, more preferably, a C₃-C₆₀ alkylgroup), a C₂-C₃₀₀ alkenyl group (preferably, a C₃-C₁₀₀ alkenyl group,more preferably, a C₃-C₆₀ alkenyl group) or a C₂-C₃₀₀ alkynyl group(preferably, a C₃-C₁₀₀ alkynyl group, more preferably, a C₃-C₆₀ alkynylgroup).

The moiety of the “aliphatic hydrocarbon group having one or morebranched chains” of the “organic group having at least one aliphatichydrocarbon group having one or more branched chains, and a total carbonnumber of not less than 14 and not more than 300” is not particularlylimited, and it may be present at the terminal (monovalent group), orother site (e.g., divalent group).

Specific examples of the “aliphatic hydrocarbon group having one or morebranched chains” include a monovalent group having one or more branchedchain(s) of a branched isomer of a propyl group, a butyl group, a pentylgroup, a hexyl group, a heptyl group, an octyl group, a nonyl group, adecyl group, an undecyl group, a dodecyl group (a lauryl group), atridecyl group, a myristyl group, a cetyl group, a stearyl group, anarachyl group, a behenyl group, an oleyl group, a linolyl group, alignoceryl group and the like, and a divalent group derived therefrom,preferably, a 3,7,11-trimethyldodecyl group, a3,7,11,15-tetramethylhexadecyl group (hereinafter sometimes to bereferred to as a 2,3-dihydrophytyl group), a2,2,4,8,10,10-hexamethylundecan-5-yl group, and the like.

When plural “aliphatic hydrocarbon groups having one or more branchedchains” are in the “organic group having at least one aliphatichydrocarbon group having one or more branched chains, and a total carbonnumber of not less than 14 and not more than 300”, each may be same ordifferent.

The moiety other than the “aliphatic hydrocarbon group having one ormore branched chains” of the “organic group having at least onealiphatic hydrocarbon group having one or more branched chains, and atotal carbon number of not less than 14 and not more than 300” can beset freely. For example, the group optionally has moieties such as —O—,—S—, —CO—, —NH—, —COO—, —OCONH—, —CONH—, —NHCO—, hydrocarbon group(monovalent group or divalent group) and the like. Examples of the“hydrocarbon group” include an aliphatic hydrocarbon group, an aromaticaliphatic hydrocarbon group, a monocyclic saturated hydrocarbon group,an aromatic hydrocarbon group and the like. Specifically, for example,monovalent groups such as an alkyl group, an alkenyl group, an alkynylgroup, a cycloalkyl group, an aryl group, an aralkyl group and the like,and divalent groups derived therefrom are used. As the “alkyl group”, aC₁₋₆ alkyl group and the like are preferable and, for example, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, hexyl and the like can be mentioned. As the “alkenyl group”, aC₂₋₆ alkenyl group and the like are preferable and, for example, vinyl,1-propenyl, allyl, isopropenyl, butenyl, isobutenyl and the like can bementioned. As the “alkynyl group”, a C₂₋₆ alkynyl group and the like arepreferable and, for example, ethynyl, propargyl, 1-propynyl and the likecan be mentioned. As the “cycloalkyl group”, a C₃₋₆ cycloalkyl group andthe like are preferable and, for example, cyclopropyl, cyclobutyl,cyclopentyl and cyclohexyl can be mentioned. As the “aryl group”, forexample, a C₆₋₁₄ aryl group and the like are preferable and, forexample, phenyl, 1-naphthyl, 2-naphthyl, biphenylyl, 2-anthryl and thelike can be mentioned. Of these, a C₆₋₁₀ aryl group is more preferable,and phenyl is particularly preferable. As the “aralkyl group”, a C₇₋₂₀aralkyl group is preferable and, for example, benzyl, 1-phenylethyl,2-phenylethyl, 1-phenylpropyl, naphthylmethyl, 1-naphthylethyl,1-naphthylpropyl and the like can be mentioned. Of these, a C₇₋₁₆aralkyl group (C₆₋₁₀ aryl-C₁₋₆ alkyl group) is more preferable, andbenzyl is particularly preferable. The “hydrocarbon group” is optionallysubstituted by a substituent selected from a halogen atom (a chlorineatom, a bromine atom, a fluorine atom, an iodine atom), an oxo group andthe like.

Z has a QR⁵ group in the number of k. Here, Q is a single bond, or —O—,—S—, —C(═O)O—, —C(═O)NH— or —NH—, preferably O. The QR⁵ group in thenumber of k may be the same or different.

In Z, the “organic group having at least one aliphatic hydrocarbon grouphaving one or more branched chains, and a total carbon number of notless than 14 and not more than 300” for R⁵ and R⁷ preferably has a totalcarbon number of not less than 14, preferably not less than 16, morepreferably not less than 18. The “organic group having at least onealiphatic hydrocarbon group having one or more branched chains, and atotal carbon number of not less than 14 and not more than 300” for R⁵and R⁷ preferably has a total carbon number of not more than 300,preferably not more than 200, more preferably not more than 160. Inaddition, in the compound of in the present invention, while a totalnumber of the branched chain of the “organic group having at least onealiphatic hydrocarbon group having one or more branched chains, and atotal carbon number of not less than 14 and not more than 300” for R⁵and R⁷ is not particularly limited, it preferably has a total number ofthe branched chain of preferably two or more, more preferably not lessthan 3, more preferably not less than 4, more preferably not less than8, more preferably not less than 10. When the total number of thebranched chain is higher, an oligonucleotide wherein the 3′-hydroxylgroup is protected by said protecting group becomes an oil showing goodsolubility in various organic solvents (particularly, non-polarsolvents) even when the oligonucleotide chain becomes long.

As the “organic group having at least one aliphatic hydrocarbon grouphaving one or more branched chains, and a total carbon number of notless than 14 and not more than 300” for R⁵ and R⁷, a group having thesame or different divalent groups represented by the formula (b):

-   wherein * shows a bonding position with the adjacent atom;-   R⁸ and R⁹ are each independently a hydrogen atom or a C₁₋₄ alkyl    group;-   X₁ is a single bond, or a C₁₋₄ alkylene group,-   provided that R⁸ and R⁹ are not hydrogen atoms at the same time, is    preferable and, for example, a group represented by any of the    following formulas (c) to (e) can be mentioned.

The carbon number, repeat unit number (m₁, n₀ to n₂) and the like in thedefinition of each symbol in the formulas (c) to (e) are shown forconvenience, and can be changed as appropriate within the range definedabove, so that the total number of the carbon will be not less than 14(preferably not less than 16, more preferably not less than 18) and notmore than 300 (preferably not more than 200, more preferably not morethan 160). In the following, the formulas (c) to (e) are successivelyexplained.

The formula (c) is as described below.

-   wherein * shows a bonding position to Q;-   R¹⁰ and R¹¹ are both hydrogen atoms, or show ═O in combination;-   n₀ is an integer of 2 to 40;-   R¹² and R¹³ in the number of n₀ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₂ in the number of n₀ are each independently a single bond or a    C₁₋₄ alkylene group;-   R¹⁴ is a hydrogen atom or a C₁₋₄ alkyl group; and-   R¹⁵ is a C₁₋₄ alkyl group;-   provided that R¹² and R¹³ are not hydrogen atoms at the same time,    and when n₀ is 2, R₁₄ is a C₁₋₄ alkyl group.

In the group of the formula (c), a group wherein

-   R¹⁰ and R¹¹ are both hydrogen atoms;-   n₀ is an integer of 2 to 40;-   R¹² and R¹³ in the number of n₀ are each independently a hydrogen    atom, a methyl group or an ethyl group;-   X₂ in the number of n₀ are each independently a single bond, a    methylene group or an ethylene group; and-   R¹⁴ is a hydrogen atom, a methyl group or an ethyl group is    preferable, provided that R¹² and R¹³ are not hydrogen atoms at the    same time, and when n₀ is 2, R¹⁴ is methyl or an ethyl group.

More preferable group of the formula (c) is a group of a branched isomerhaving a carbon number of 14 to 160, of a myristyl group, a cetyl group,a stearyl group, an arachyl group, a behenyl group and the like. Ofthese, a 2,3-dihydrophytyl group, a 3,7,11-trimethyldodecyl group and a2,2,4,8,10,10-hexamethyl-5-dodecanoyl group are particularly preferable.

The formula (d) is as described below.

-   wherein * shows a bonding position to Q;-   OR¹⁶ in the number of m₁ are each independently hydroxyl group    substituted by a group represented by the formula (c); and-   m₁ is an integer of 1 to 3.

The group represented by the formula (c) is the same as the grouprepresented by the above-mentioned formula (c) except that * does notshow a bonding position to Q but shows a bonding position to O.

In the group of the formula (d), R¹⁶ is more preferably a group of abranched isomer having a carbon number of 14 to 30 of a myristyl group,a cetyl group, a stearyl group, an arachyl group, a behenyl group andthe like. Of these, a 2,3-dihydrophytyl group and a3,7,11-trimethyldodecyl group are particularly preferable.

The formula (e) is as described below.

-   wherein * shows a bonding position to Q;-   n₁ is an integer of 1 to 10;-   n₂ is an integer of 1 to 10;-   R²⁰ and R²¹ in the number of n₁ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₃ in the number of n₁ are each independently a single bond or a    C₁₋₄ alkylene group;-   R²² and R²³ in the number of n₂ are each independently a hydrogen    atom or a C₁₋₄ alkyl group;-   X₅ in the number of n₂ are each independently a single bond or a    C₁₋₄ alkylene group;-   X₄ is a single bond or a C₁₋₄ alkylene group; and-   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵ and R²⁶ are each independently a hydrogen    atom or a C₁₋₄ alkyl group,-   provided that R²⁰ and R²¹ and/or R²² and R²³ are not hydrogen atoms    at the same time, and when n₁+n₂ is 2, two or more of R¹⁷, R¹⁸ and    R¹⁹ are each independently a C₁₋₄ alkyl group, or two or more of    R²⁴, R²⁵ and R²⁶ are each independently a C₁₋₄ alkyl group.

A group of the formula (e), wherein

-   n₁ is an integer of 1 to 5;-   n₂ is an integer of 1 to 5;-   R²⁰ and R²¹ in the number of n₁ are each independently a hydrogen    atom, a methyl group or an ethyl group;-   X₃ in the number of n₁ are each independently a single bond, a    methylene group or an ethylene group;-   R²² and R²³ in the number of n₂ are each independently a hydrogen    atom, a methyl group or an ethyl group;-   X₅ in the number of n₂ are each independently a single bond, a    methylene group or an ethylene group;-   X₄ is a single bond, a methylene group or an ethylene group; and-   R¹⁷, R¹⁸, R¹⁹, R²⁴, R²⁵ and R²⁶ are each independently a hydrogen    atom or a C₁₋₄ alkyl group is more preferable,-   provided that R²⁰ and R²¹ and/or R²² and R²³ are not hydrogen atoms    at the same time, and when n₁+n₂ is 2, two or more of R¹⁷, R¹⁸ and    R¹⁹ are each independently a C₁₋₄ alkyl group, or two or more of    R²⁴, R²⁵ and R²⁶ are each independently a C₁₋₄ alkyl group.

A group of the formula (e), wherein

-   n₁ is an integer of 1 to 5;-   n₂ is an integer of 1 to 5;-   R²⁰ and R²¹ in the number of n₁ are each independently a hydrogen    atom or a methyl group;-   X₃ in the number of n₁ are each independently a single bond or a    methylene group;-   R²² and R²³ in the number of n₂ are each independently a hydrogen    atom or a methyl group;-   X₅ in the number of n₂ are each independently a single bond or a    methylene group;-   X₄ is a single bond or a methylene group; and-   R¹⁷, R¹⁸, R¹⁹, R²⁵, R²⁵ and R²⁶ are each a methyl group, provided    that R²⁰ and R²¹ and/or R²² and R²³ are not hydrogen atoms at the    same time is particularly preferable.

Specific examples of the “organic group having at least one aliphatichydrocarbon group having one or more branched chains, and a total carbonnumber of not less than 14 and not more than 300” for R⁵ and R⁷ includethe following groups, wherein * in each group shows a bonding position,n₃ in the formula is an integer of not less than 3, and n₄ can beappropriately adjusted so that the total carbon number of the group willbe not less than 14 and not more than 300.

Specific preferable examples of the “organic group having at least onealiphatic hydrocarbon group having one or more branched chains, and atotal carbon number of not less than 14 and not more than 300” for R⁵and R⁷ include the following groups:

-   3,7,11,15-tetramethylhexadecyl group;-   3,7,11-trimethyldodecyl group;-   2,2,4,8,10,10-hexamethyl-5-dodecanoyl group;-   3,4,5-tri(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group; and-   3,5-di(3′,7′,11′,15′-tetramethylhexadecyloxy)benzyl group.

Preferable examples of protecting group represented by the formula(III′) or the formula (III) of the present invention include thefollowing benzylsuccinyl group, or diphenylmethylsuccinyl group, whichare not to be construed as limiting the present invention:

-   2-{2,4-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group; 3,5-di(2′,3′-dihydrophytyloxy)benzylsuccinyl group;    4-(2′,3′-dihydrophytyloxy)benzylsuccinyl group;    2-{1-[(2-chloro-5-(2′,3′-dihydrophytyloxy)phenyl)]benzylaminocarbonyl}ethylcarbonyl    group; 3,4,5-tri(2′,3′-dihydrophytyloxy)benzylsuccinyl group;    2-{3,4,5-tri(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;    2-{4-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;    2-{2-[3′,4′,5′-tri(2″,3″-dihydrophytyloxy)benzyloxy]-4-methoxybenzylaminocarbonyl}ethylcarbonyl    group;    2-{4-(2′,3′-dihydrophytyloxy)-2-methoxybenzylaminocarbonyl}ethylcarbonyl    group; 4-(2′,3′-dihydrophytyloxy)-2-methylbenzylsuccinyl group;    2-{4-(2′,3′-dihydrophytyloxy)-2-methylbenzylaminocarbonyl}ethylcarbonyl    group; 4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylsuccinyl    group;    2-{4-[2,2,4,8,10,10-hexamethyl-5-dodecanoylamino]benzylaminocarbonyl}ethylcarbonyl    group; 4-(3,7,11-trimethyldodecyloxy)benzylsuccinyl group;    2-{4-(3,7,11-trimethyldodecyloxy)benzylaminocarbonyl}ethylcarbonyl    group;    2-{3,5-di(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;    2-{1-[2,3,4-tri(2′,3′-dihydrophytyloxy)phenyl]benzylaminocarbonyl}ethylcarbonyl    group;    2-{1-[4-(2′,3′-dihydrophytyloxy)phenyl]-4′-(2′,3′-dihydrophytyloxy)benzylaminocarbonyl}ethylcarbonyl    group;    3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylsuccinyl    group; and    2-{3,4,5-tris[3,4,5-tri(2′,3′-dihydrophytyloxy)benzyl]benzylaminocarbonyl}ethylcarbonyl    group.    4-3. Explanation of “Optionally Protected Nucleic Acid Base”

The “optionally protected nucleic acid base” represented by Base in theformulas (i) and (ii) means, for example, that an amino group may beprotected in an adenyl group, a guanyl group or a cytosyl group, whichis a nucleic acid base having an amino group, or an imido group may beprotected in a thymyl group, or an uracil group, which is a nucleic acidbase having a cyclic imido group, a nucleic acid base wherein the aminogroup therein is protected by a protecting group sustainable under thedeprotection conditions of the 5′-position is preferable. The“amino-protecting group” and “imide-protecting group” are notparticularly limited, and examples thereof include the protecting groupsdescribed in Greene's PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, 4thedition, JOHN WILLY&SONS, 2006 and the like. Specific examples of the“amino-protecting group” include a pivaloyl group, a pivaloyloxymethylgroup, a trifluoroacetyl group, a phenoxyacetyl group, a4-isopropylphenoxyacetyl group, a 4-tert-butylphenoxyacetyl group, anacetyl group, a benzoyl group, an isobutyryl group, adimethylformamidinyl group, a 9-fluorenylmethyloxycarbonyl group and thelike. Among them, a phenoxyacetyl group, a 4-isopropylphenoxyacetylgroup, an acetyl group, a benzoyl group, an isobutyryl group and adimethylformamidinyl group are preferable. In addition, the carbonylgroup of the nucleic acid base is optionally protected, and can beprotected, for example, by reacting phenol, 2,5-dichlorophenol,3-chlorophenol, 3,5-dichlorophenol, 2-formylphenol, 2-naphthol,4-methoxyphenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol,4-acetylaminophenol, pentafluorophenol, 4-pivaloyloxybenzyl alcohol,4-nitrophenethyl alcohol, 2-(methylsulfonyl)ethanol,2-(phenylsulfonyl)ethanol, 2-cyanoethanol, 2-(trimethylsilyl)ethanol,dimethylcarbamoyl chloride, diethylcarbamoyl chloride,ethylphenylcarbamoyl chloride, 1-pyrrolidinecarbonyl chloride,4-morpholinecarbonyl chloride, diphenylcarbamoyl chloride and the'like.In some cases, the carbonyl-protecting group does not need to beparticularly introduced.

Another preferable embodiment of the protecting group of the nucleicacid base includes a “group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ straight chain or branched chainalkenyl group”. The “group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ straight chain or branched chainalkenyl group” is as defined for the “group having a C₅₋₃₀ straightchain or branched chain alkyl group and/or a C₅₋₃₀ straight chain orbranched chain alkenyl group” of the “nucleic acid base protected by agroup having a C₅₋₃₀ straight chain or brandied chain alkyl group and/ora C₅₋₃₀ straight chain or branched chain alkenyl group” for Base² in theabove-mentioned formula (I).

Of these, a “group having a C₅₋₃₀ branched chain alkyl group and/or aC₅₋₃₀ branched chain alkenyl group” is preferable, and a “group having aC₅₋₃₀ branched chain alkyl group” is more preferable.

Protection of a nucleic acid base by a “group having a C₅₋₃₀ straightchain or branched chain alkyl group and/or a C₅₋₃₀ straight chain orbranched chain alkenyl group” further confers liposolubility andsolubility in an organic solvent (particularly, non-polar solvent) to ann-mer oligonucleotide, which is advantageous for the synthesis of a longchain oligonucleotide.

At least one nucleic acid base of an n-mer oligonucleotide is preferablyprotected by a group having a C₅₋₃₀ straight chain or branched chainalkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenylgroup, and more preferably protected by a group having a C₅₋₃₀ branchedchain alkyl group and/or a C₅₋₃₀ branched chain alkenyl group.

In this case, at least one nucleic acid base of the n-meroligonucleotide only needs to be protected by the protecting group, orall nucleic acid bases in the number of n may be is protected by theprotecting group, or a part thereof may be protected by the protectinggroup and other nucleic acid bases may be protected by a protectinggroup conventionally used in the field of nucleic acid synthesis (e.g.,pivaloyl group, pivaloyloxymethyl group, trifluoroacetyl group,phenoxyacetyl group, 4-isopropylphenoxyacetyl group,4-tert-butylphenoxyacetyl group, acetyl group, benzoyl group, isobutyrylgroup, dimethylformamidinyl group, 9-fluorenylmethyloxycarbonyl groupetc.).

Thus, when all or a part of the nucleic acid bases represented by Baseare/is protected by a group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ straight chain or branched chainalkenyl group, the obtained n+p-mer oligonucleotide shows furtherimproved liposolubility and solubility in an organic solvent(particularly, non-polar solvent), which facilitates the extractionoperation in the next step (4) and enables synthesis of anoligonucleotide having a higher degree of polymerization.

In the nucleic acid base in the number of n, the ratio of protection bya group having a C₅₋₃₀ straight chain or branched chain alkyl groupand/or a C₅₋₃₀ straight chain or branched chain alkenyl group can beappropriately set so that the n-mer oligonucleotide shows sufficientsolubility in an organic solvent (particularly, non-polar solvent).

Preferable examples of the “group having a C₅₋₃₀ straight chain orbranched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group” and “group having a C₅₋₃₀ branched chain alkylgroup and/or a C₅₋₃₀ branched chain alkenyl group” are as explained forthe aforementioned formula (I).

4-4. Oligonucleotide Protected by a Branched Chain-Containing AromaticGroup

A more preferable embodiment of the n-mer oligonucleotide in the presentinvention is a novel oligonucleotide represented by the followingformula (II), wherein, in the aforementioned formulas (i) and (ii), a3′-hydroxyl-protecting group represented by P³ is a group represented bythe aforementioned formula (III): -L-Y—Z (sometimes to be referred to as“branched chain-containing aromatic protecting group” in the presentspecification) (sometimes to be referred to as an “oligonucleotideprotected by a branched chain-containing aromatic group” in the presentspecification).

When m is 0, the oligonucleotide protected by a branchedchain-containing aromatic group, which is represented by the formula(II), is understood to mean a “nucleoside protected by a branchedchain-containing aromatic group”.

Since the oligonucleotide protected by a branched chain-containingaromatic group of the present invention is easily soluble in a non-polarsolvent superior in the partitioning operability, which is a reactionsolvent in the production method of the present invention, it isextremely useful as a novel compound usable for a production method ofoligonucleotide wherein the reaction in each step can be performedsmoothly, and the final resultant product can be obtained withoutcrystallization and isolation of each intermediate but via an extractionseparation alone (also referred to as one-pot synthesis method).

The compound should be clearly differentiated from the protecting grouphaving a straight chain structure described in JP-A-2010-275254 since itshows good solubility particularly in heptane as a representativesolvent of non-polar solvents.

In addition, as compared to conventional liquid phase methods, since thecompound permits stable dissolution and transfer into a nonpolar solventirrespective of the degree of polymerization (sequence and chain length)of oligonucleotide, it is advantageous in that the isolation andpurification step can be simplified as for the steps, and high purityand high yield can be ensured as a total view.

While the lower limit of the solubility (=solute/(solvent+solute)×100)(mass %) of a nucleoside protected by a branched chain-containingaromatic group wherein m is 0 in heptane at 20° C. is not particularlylimited as long as the binding to a reaction substrate and the reactionthereafter proceed, it is preferably 1 mass %, more preferably 2 mass %,further preferably 5 mass %, still more preferably 10 mass %, especiallypreferably 25 mass %, particularly preferably 50 mass %.

The upper limit of the solubility (=solute/(solvent+solute)×100) (mass%) of a nucleoside protected by a branched chain-containing aromaticgroup wherein m is 0 in heptane at 20° C. is preferably 80 mass %, morepreferably 85 mass %, further preferably 90 mass %, still morepreferably 95 mass %, particularly preferably 98 mass %, since thereaction can proceed stably irrespective of the industrial progressdegree of the reaction.

In the present specification, the “solubility” means the percentage(mass %) of the mass of solute relative to the total mass of the solventand the solute when the solute is saturated in the solvent.

The formula (II):

wherein

-   m is an integer of 0 or more;-   Base¹ in the number of m+1 are each independently an optionally    protected nucleic acid base;-   P¹ is a hydrogen atom, or a temporary protecting group removable    under acidic conditions;-   X is a hydrogen atom, an optionally protected hydroxyl group, a    halogen atom or an organic group crosslinked with the 4-position    carbon atom;-   X′ in the number of m are each independently a hydrogen atom, an    optionally protected hydroxyl group, a halogen atom or an organic    group crosslinked with the 4-position carbon atom;-   P² in the number of m are each independently a protecting group    removable under basic conditions;-   R³⁴ in the number of m are each independently an oxygen atom or a    sulfur atom; and-   L, Y and Z are as defined above.

In compound (II) of the present invention, p-mer oligonucleotide whereinthe 3′-hydroxyl group is phosphoramidited and the 5′-hydroxyl group isprotected by a temporary protecting group is bonded via oxygen atom of5′-hydroxyl group to form m+1+p-mer oligonucleotide.

The compound (II) wherein m is 0 of the present invention is a startingcompound of 3′-terminal in the oligonucleotide synthesis. In addition,the compound of the present invention also encompasses a compoundwherein 5′-hydroxyl group is not protected (P¹ is a hydrogen atom) in awide sense.

The definition, preferable embodiments and the like of P¹, P², X, X′ andR³⁴ are similar to those of the above-mentioned compound (I).

The definition, preferable embodiments and the like of m are similar tothose of the above-mentioned compound (i) and (ii).

The preferable embodiments of L, Y and Z in the formula (II) are similarto those of L, Y and Z in the aforementioned formula (III′) or (III).

The “optionally protected nucleic acid base” represented by Base¹ issimilar to that in the aforementioned formulas (i) and (ii).

Since the oligonucleotide protected by a branched chain-containingaromatic group, which is represented by the formula (II), is conferredwith sufficient liposolubility by a branched chain-containing aromaticprotecting group, it is not necessary; however, Base¹ is optionallyprotected by a group having a C₅₋₃₀ straight chain or branched chainalkyl group and/or a C₅₋₃₀ straight chain or branched chain alkenylgroup, preferably a group having a C₅₋₃₀ branched chain alkyl groupand/or a C₅₋₃₀ branched chain alkenyl group to further improve theliposolubility.

Preferable examples of the “group having a C₅₋₃₀ straight chain orbranched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group” and the “group having a C₅₋₃₀ branched chain alkylgroup and/or a C₅₋₃₀ branched chain alkenyl group” are as mentionedabove.

A preferable embodiment of the compound represented by the formula (II)of the present invention is a compound of the formula (IIa), wherein

-   m is 0;-   Base¹ is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected;-   P¹ is a di (C₁₋₆ alkoxy)trityl group, or a mono (C₁₋₆ alkoxy)trityl    group;-   X is a hydrogen atom, an optionally protected hydroxyl group,    fluorine atom, —ORi (Ri is as defined above), —O—NR³⁷—Rj (Rj and R³⁷    are as defined above), or —O—Rk-C—Rl (Rk and Rl are as defined    above); and-   L-Y—Z is the combination of each group shown as a preferable    embodiment in the aforementioned formula (III′) or the formula    (III).

Another preferable embodiment of the compound represented by the formula(II) of the present invention is a compound of the formula (IIb),wherein

-   m is 0;-   Base¹ is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected;-   P¹ is a dimethoxytrityl group or a monomethoxytrityl group;-   X is a hydrogen atom, an optionally protected hydroxyl group,    fluorine atom, —O—CH₂—, —O—CH₂—CH₂—, or —O—NR³⁷—CH₂— (R³⁷ is as    defined above), —O—CH₂—O—CH₂— (in all of which the left side binds    to the 2-position and the right side binds to the 4-position); and-   L-Y—Z is the combination of each group shown as a preferable    embodiment in the aforementioned formula (III′) or the formula    (III).

A still another preferable embodiment of the compound represented by theformula (II) of the present invention is a compound of the formula(IIc), wherein

-   m is 0;-   Base¹ is a cytosyl group, a uracil group, a thyminyl group, an    adenyl group, or a guanyl group, each of which is optionally    protected;-   P¹ is a dimethoxytrityl group;-   X is a hydrogen atom, methoxy group, tert-butyldimethylsilyloxy    group, fluorine atom, —O—CH₂—, —O—CH₂—CH₂—, —O—NH—CH₂—, —O—NMe-CH₂—,    —O—CH₂—O—CH₂— (in all of which the left side binds to the 2-position    and the right side binds to the 4-position); and L-Y—Z is the    combination of each group shown as a preferable embodiment in the    aforementioned formula (III′) or the formula (III).    4-5. Production Method of Oligonucleotide Protected by Branched    Chain-Containing Aromatic Group

While the production method of a nucleoside protected by a branchedchain-containing aromatic group represented by the formula (II′), whichis an oligonucleotide protected by a branched chain-containing aromaticgroup represented by the formula (II) wherein m is 0, is notparticularly limited, it can be produced by a method known per se(Richard T. Pon et al., Nucleic Acids Research 2004, 32, 623-631) or amethod analogous thereto.

When a starting compound has a substituent (e.g., hydroxyl group, aminogroup, carboxy group) that influences the reaction, the startingcompound is generally protected in advance by a suitable protectinggroup according to a known method and then subjected to the reaction.Such protecting group can be removed after the reaction by a knownmethod such as an acid treatment, an alkali treatment, a catalyticreduction and the like.

A general production method of a nucleoside protected by a branchedchain-containing aromatic group of the above-mentioned formula (II′)wherein L is a succinyl group is shown below.

wherein each symbol is as defined above.

Nucleoside (q) wherein 5′-hydroxyl group is protected by protectinggroup P¹ is reacted with succinic anhydride in the presence of a base togive compound (r) wherein succinic acid is introduced into 3′-hydroxylgroup. A nucleoside protected by a branched chain-containing aromaticgroup represented by the formula (II′) can be obtained by dehydrationcondensation of compound (r) with Z—Y—H in the presence of a condensingagent.

The conversion step of the above-mentioned nucleoside (q) to compound(r) is advantageously performed in a solvent inert to the reaction.While such solvent is not particularly limited as long as the reactionproceeds, halogenated hydrocarbon solvents such as dichloromethane,1,2-dichloroethane, chloroform, carbon tetrachloride and the like,aromatic hydrocarbon solvents such as benzene, toluene, xylene and thelike, aliphatic hydrocarbon solvents such as pentane, hexane, heptane,octane and the like, ether solvents such as diethyl ether,tetrahydrofuran, cyclopentyl methyl ether and the like, and mixedsolvents thereof are preferable. Of these, dichloromethane andchloroform are particularly preferable.

While the base is not particularly limited, for example, an organic basementioned below can be used, with preference given to triethylamine.

The above-mentioned dehydrating condensation step is advantageouslyperformed in a solvent inert to the reaction. While such solvent is notparticularly limited as long as the reaction proceeds, halogenatedhydrocarbon solvents such as dichloromethane, 1,2-dichloroethane,chloroform, carbon tetrachloride and the like, aromatic hydrocarbonsolvents such as benzene, toluene, xylene and the like, or aliphatichydrocarbon solvents such as pentane, hexane, heptane, octane and thelike, and mixed solvents thereof are preferable. Of these,dichloromethane and chloroform are particularly preferable.

Examples of the condensing agent used for the condensation reaction ofcompound (r) with Z—Y—H include dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),N-ethyl-N′-3-dimethylaminopropylcarbodiimide and hydrochloride thereof(EDC HCl), (benzotriazol-1-yloxy)tripyrrolidinophosphoniumhexafluorophosphate (PyBop),O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU),1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium-3-oxidehexafluorophosphate (HCTU), O-benzotriazole-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU) and the like. Of these, HBTU, HCTU,N-ethyl-N′-3-dimethylaminopropylcarbodiimide and hydrochloride thereof(EDC HCl) are preferable.

The amount of the condensing agent to be used is 1 to 10 mol, preferably1 to 5 mol, per 1 mol of compound (r). The amount of Z—Y—H to be used is1 to 10 mol, preferably 1 to 5 mol, per 1 mol of compound (r). While thereaction temperature is not particularly limited as long as the reactionproceeds, it is preferably −10° C. to 50° C., more preferably 0° C. to30° C. The reaction time is 30 min to 70 hr.

A compound of the above-mentioned formula (II′) wherein 1 is other thana succinyl group can also be produced by performing a reaction similarto the above-mentioned production method except that a correspondingacid anhydride, a corresponding dicarboxylic halide, an activated esterof corresponding dicarboxylic acid and the like is used instead ofsuccinic anhydride.

A compound of the above-mentioned formula (II) wherein m is one or morecan be produced by repeating the 5′-terminal elongation processaccording to the following production method of the present inventionand using a compound represented by the formula (II′) as startingmaterial.

4-6. Production Method of Z—Y—H (Alcohol or Amine)

While the production method of an alcohol compound or an amine compoundrepresented by the formula: Z—Y—H, which is a starting compound used forthe production of an oligonucleotide protected by a branchedchain-containing aromatic group, is not particularly limited, forexample, it can be produced by the following steps.

-   wherein Q′ is —O—, —S—, —C(═O)O— or —NH—, R_(g) is a hydrogen atom,    an OR_(h) group (wherein R_(h) is an alkyl group such as a C₁₋₆    alkyl group and the like, an aralkyl group such as benzyl group and    the like, and the like) or a group represented by the formula (a3):

-   wherein each symbol is as defined above, Y₁ is a leaving group such    as a halogen atom and the like, and other symbol is as defined    above.    Step (a)

In this step, an R⁵ group is introduced into a Q′H group wherein Q′ is—O—, —S—, —C(═O)O— or —NH— of a compound represented by the formula (IV)(hereinafter to be abbreviated as compound (IV)) to give a compoundrepresented by the formula (IVa) (hereinafter to be abbreviated ascompound (IVa)).

When Q′ is —O—, —S— or —NH—, the reaction is carried out in a solventthat does not influence the reaction, in the presence or absence of abase and using a halide corresponding to an R⁵ group (chloride, bromideor iodide), a carboxylic acid or an acid halide corresponding to an R⁵group or alkylsulfonyloxylation product (e.g., methanesulfonyloxylationproduct etc.) or an arylsulfonyloxylation product (e.g.,p-toluenesulfonyloxylation product etc.) corresponding to an R⁵ group.In addition, when Q′ is —O—, the reaction can be carried out under theconditions of Mitsunobu reaction including reacting compound (IV) withhydroxide corresponding to an R⁵ group in the presence oftriphenylphosphine and diisopropyl azodicarboxylate. Furthermore, whenQ′ is —C(═O)O—, for example, compound (IVa) can be synthesized byreacting compound (IV) with amine or hydroxide corresponding to an R⁵group in the presence of the below-mentioned condensing agent.

Examples of the base include alkali metal salt such as sodium carbonate,sodium hydrogen carbonate, potassium carbonate, sodium hydride,potassium hydride, potassium tert-butoxide and the like; amines such aspyridine, triethylamine, N,N-dimethylaniline,1,8-diazabicyclo[5.4.0]undec-7-ene etc., and the like. Of these, sodiumcarbonate, potassium carbonate, sodium hydride and the like arepreferable.

Examples of the solvent include aromatic hydrocarbons such as toluene,xylene and the like; ethers such as tetrahydrofuran, dioxane and thelike; amides such as dimethylformamide, dimethylacetamide and the like;halogenated hydrocarbons such as chloroform, dichloromethane and thelike; nitriles such as acetonitrile and the like, N-methylpyrrolidone,and a mixture thereof. Of these, dimethylformamide, tetrahydrofuran,toluene, N-methylpyrrolidone and the like are preferable.

The reaction temperature is preferably 50° C. to 150° C., morepreferably 60° C. to 130° C. The reaction time is preferably 2 to 30 hr,more preferably 3 to 10 hr.

Step (b)

In this step, compound (IVa) is reduced to give a compound representedby the formula (I-a) (hereinafter to be abbreviated as compound (I-a)).The reduction reaction can be performed by a method using a reducingagent.

Examples of the reducing agent to be used for the reduction reactioninclude metal hydride (sodium borohydride, lithium borohydride, sodiumcyanoborohydride, sodium triacetoxyborohydride, dibutylaluminum hydride,aluminum hydride, lithium aluminum hydride, etc.) and the like. Ofthese, sodium borohydride, dibutylaluminum hydride and the like arepreferable.

The reaction is performed in a solvent that does not influence thereaction. Examples of the solvent include alcohols such as methanol,ethanol and the like; ethers such as diethyl ether, tetrahydrofuran,dioxane and the like; aromatic hydrocarbons such as toluene, xylene andthe like; and a mixture thereof. Of these, tetrahydrofuran, toluene andthe like are preferable.

The reaction temperature is preferably 0° C. to 100° C., more preferably30° C. to 70° C., and the reaction time is preferably 1 to 24 hr, morepreferably 2 to 5 hr.

Step (c)

In this step, compound (IVa) (in the formula (IVa), R_(g) is not ahydrogen atom or an OR_(h), group) is reduced in the same manner as inthe above-mentioned step (b).

Step (d-1)

In this step, compound (IVa) (in the formula (IVa), Rg is a hydrogenatom) is oximated to give a compound represented by the formula (I′-a)(hereinafter to be abbreviated as compound (I′-a)).

The oximation reaction includes reacting compound (IVa) withhydroxylamine acid addition salt in a solvent that does not influencethe reaction in the presence of a base.

Examples of the hydroxylamine acid addition salt include mineral acidsalts such as hydrochloride, sulfate, nitrate and the like, organic acidsalts such as acetate, trifluoroacetate, methanesulfonate,trifluoromethanesulfonate, p-toluenesulfonate etc., and the like, andhydrochloride is particularly preferable.

Examples of such base include alkali metal salts such as potassiumhydroxide, sodium hydroxide, sodium hydrogen carbonate, potassiumcarbonate and the like; organic amines such as pyridine, triethylamine,diisopropylethylamine, N,N-dimethylaniline,1,8-diazabicyclo[5.4.0]undec-7-ene etc., and the like. Of these,triethylamine, diisopropylethylamine and the like are preferable.

Examples of the solvent include halogen solvents such as chloroform,dichloromethane and the like; aromatic hydrocarbons such as toluene,xylene and the like; ethers such as tetrahydrofuran, dioxane and thelike; and/or a mixture thereof. Of these, dichloromethane, chloroform,toluene and the like are preferable.

The reaction temperature is preferably 10° C. to 100° C., morepreferably 20° C. to 60° C., and the reaction time is preferably 0.5 to30 hr, more preferably 2 to 20 hr.

Step (d-2)

In this step, compound (I′-a) is reduced by a catalytic hydrogenationreaction in the presence of a metal catalyst such as palladium-carbon,Raney-nickel and the like, or by a reducing agent such as metal hydrideand the like, which is similar to those in the aforementioned step (b),to give a compound represented by the formula (I-b) (hereinafter to beabbreviated as compound (I-b)), which is the compound of the presentinvention.

Compound (I-b) can also be produced from step (d-3) via step (d-4) andstep (d-5).

Step (d-3)

In this step, compound (I-a) is halogenated with, for example, achlorinating agent such as acetyl chloride, thionyl chloride and thelike or, for example, a brominating agent such as acetyl bromide,phosphorus tribromide, diphenylphosphine/bromine and the like to give acompound represented by the formula (I′-b) (hereinafter to beabbreviated as compound (I′-b)).

Examples of the solvent include halogenated hydrocarbons such aschloroform, dichloromethane, and the like; aromatic hydrocarbons such astoluene, xylene, and the like; ethers such as tetrahydrofuran, dioxane,and the like; and a mixture thereof. Of these, chloroform,tetrahydrofuran, toluene, and the like are preferable.

The reaction temperature is preferably 10° C. to 150° C., morepreferably 30° C. to 80° C., and the reaction time is preferably 0.5 to30 hr, more preferably 2 to 20 hr.

Step (d-4)

In this step, compound (I′-b) is azidated with an azidating agent suchas sodium azide and the like to give a compound represented by theformula (I′-c) (hereinafter to be abbreviated as compound (I′-c)).

The reaction includes reacting compound (I′-b) with an azidating agentin a solvent that does not influence the reaction.

Examples of the solvent include halogenated hydrocarbons such aschloroform, dichloromethane, and the like; aromatic hydrocarbons such astoluene, xylene, and the like; ethers such as tetrahydrofuran, dioxane,and the like; amides such as N,N-dimethylformamide and the like; and amixture thereof. Of these, chloroform, N,N-dimethylformamide, and thelike are preferable.

The reaction temperature is preferably 10° C. to 150° C., morepreferably 20° C. to 100° C., and the reaction time is preferably 0.5 to30 hr, more preferably 2 to 20 hr.

Step (d-5)

In this step, compound (I′-c) is aminated to give compound (I-b).

The reaction is carried out by reacting compound (I′-c) withtriphenylphosphine in a solvent that does not influence the reaction inthe presence of water or catalytic hydrogenation.

The amount of triphenylphosphine to be used is preferably 1 to 10 mol,particularly preferably 1 to 5 mol, per 1 mol of compound (I′-c).

The amount of water to be used is preferably 1 to 10 mol, particularlypreferably 1 to 5 mol, per 1 mol of compound (I′-c).

Examples of the solvent include aromatic hydrocarbons such as toluene,xylene, and the like; ethers such as tetrahydrofuran, dioxane, and thelike; and a mixture thereof. Of these, toluene, tetrahydrofuran, and thelike are preferable.

The reaction temperature is preferably 10° C. to 150° C., morepreferably 20° C. to 100° C., and the reaction time is preferably 0.5 to30 hr, more preferably 2 to 20 hr.

Step (d-6)

In this step, compound (I′-b) is reacted with RNH₂ (wherein R is asdefined above) to give a compound represented by the formula (I-c)(hereinafter to be abbreviated as compound (I-c)), which is the compoundof the present invention wherein Y is an —NHR group.

The reaction includes reacting compound (I′-b) with amine represented byR—NH₂ in a solvent that does not influence the reaction in the presenceof, where necessary, for example, a base such as tertiary amine(triethylamine, diisopropylethylamine etc.) and the like.

Examples of the solvent include aromatic hydrocarbons such as toluene,xylene, and the like; ethers such as tetrahydrofuran, dioxane, and thelike; and, halogen solvents such as chloroform, dichloromethane, and thelike and a mixture thereof. Of these, toluene, tetrahydrofuran,chloroform, and the like are preferable.

The reaction temperature is generally 10° C. to 100° C., preferably 20°C. to 60° C., and the reaction time is generally 0.5 to 30 hr,preferably 2 to 20 hr.

Step (d-7)

In this step, compound (I-d) is reacted with a compound having a —CONH₂group or a —OCONH₂ group, and treated with a base to give compound(I-e).

The reaction of compound (I-d) with a compound having a —CONH₂ group ora —OCONH₂ group is carried out in a solvent that does not influence thereaction and under an acid catalyst.

Examples of the acid catalyst include methanesulfonic acid,trifluoromethanesulfonic acid, toluenesulfonic acid and the like. Ofthese, methanesulfonic acid and toluenesulfonic acid are preferable.

The amount of the acid catalyst to be used is preferably 0.05 to 0.5mol, particularly preferably 0.1 to 0.3 mol, per 1 mol of compound(I-d).

Examples of the compound having a —CONH₂ group or a —OCONH₂ groupinclude Fmoc-NH₂, HCONH₂, CF₃CONH₂, AcNH₂, EtOCONH₂, Cbz-NH₂ and thelike. Of these, Fmoc-NH₂, EtOCONH₂ and the like are preferable.

Here, the “Fmoc-” means a 9-fluorenylmethoxycarbonyl group (hereinafterto be also referred to as a Fmoc group), and “Cbz-” means abenzyloxycarbonyl group (hereinafter to be also referred to as a Cbzgroup).

The R⁵ forming-reagent to be used as a starting compound of step (a)[i.e., hydroxide, halide, an alkylsulfonyloxylation product (e.g.,methanesulfonyloxylation product etc.) or an arylsulfonyloxylationproduct (e.g., p-toluenesulfonyloxylation product etc.) corresponding toR⁵ group] may be a commercially available product. In addition, the R⁵forming-reagent can be produced by, for example,

-   (1) halogenation, alkylsulfonyloxylation or arylsulfonyloxylation of    hydroxide corresponding to an R⁵ group, or-   (2) reduction reaction of unsaturated hydroxide corresponding to an    R⁵ group (e.g., catalytic hydrogenation reaction in the presence of    a metal catalyst such as platinum-carbon (Pt/C), palladium-carbon    (Pd/C), rhodium-carbon (Rh/C), Raney-nickel etc. and the like), and    subsequently halogenation, alkylsulfonyloxylation or    arylsulfonyloxylation.

In the production of the R⁵ forming-reagent, examples of the reagent tobe used for conversion to a leaving group from a hydroxyl group include,in addition to halogenating agent such as chlorinating agent (thionylchloride, N-chlorosuccinimide (NCS) and the like), brominating agent(hydrobromic acid, acetyl bromide, N-bromosuccinimide (NBS), phosphorustribromide, diphenylphosphine/bromine and the like) and the like,alkylsulfonylating agent such as methanesulfonyl chloride,trifluoromethanesulfonyl chloride and the like, arylsulfonylating agentsuch as benzenesulfonyl chloride, p-toluenesulfonyl chloride etc. andthe like. Of these, thionyl chloride, hydrobromic acid and the like arepreferable, which are the halogenating agents.

The reaction is performed in a solvent that does not influence thereaction. Examples of the solvent include water, halogenatedhydrocarbons such as chloroform, dichloromethane and the like; aromatichydrocarbons such as benzene, toluene, xylene and the like; nitrilessuch as acetonitrile, propionitrile and the like; ethers such astetrahydrofuran, 1,4-dioxane, diethyl ether and the like. Of these,water, halogenated hydrocarbons such as chloroform and the like arepreferable.

The reaction temperature is preferably 10 to 120° C., more preferably 50to 100° C., and the reaction time is preferably 1 to 72 hr, morepreferably 3 to 24 hr.

The compound represented by Z—Y—H wherein the aforementioned Q is asingle bond can be also produced by, for example, the following method.That is, introduction of an R⁵ group onto a benzene ring can be carriedout by

-   (1) Friedel-Crafts reaction using halide corresponding to an R⁵    group (chloride, bromide, or iodide), carboxylic acid or acid halide    corresponding to an R⁵ group,-   (2) a method comprising subjecting a compound corresponding to the    above-mentioned compound (II) (a compound wherein a Q′H group is    substituted by a —CHO group) to carbon homologation by a Wittig    reaction and, followed by catalytic hydrogenation and the like, or-   (3) conventional organic synthesis reaction such as cross coupling    using a metal catalyst and the like.

In each scheme above, the carbon number of an organic group for R⁵, thekind of halogen atom, reaction reagents and the like are shown for thesake of convenience, and can be appropriately changed within the scopeof the above-mentioned definitions.

4-7. Explanation of “Oligonucleotide Comprising a Protected Base Whereinthe 3′-hydroxyl Group is Phosphoramidited, the 5′-hydroxyl Group isProtected by a Temporary Protecting Group Removable Under AcidicConditions, and the Nucleic Acid Base is Protected by a Group Having aC₅₋₃₀ Straight Chain or Branched Chain Alkyl Group and/or a C₅₋₃₀Straight Chain or Branched Chain Alkenyl Group”

The “p-mer oligonucleotide comprising a protected base (p is an integerof one or more) wherein the 3′-hydroxyl group is phosphoramidited, the5′-hydroxyl group is protected by a temporary protecting group removableunder acidic conditions, and the nucleic acid base is protected by agroup having a C₅₋₃₀ straight chain or branched chain alkyl group and/ora C₅₋₃₀ straight chain or branched chain alkenyl group” used in step (2)is not particularly limited as long as it satisfies the structurerequirements.

“3′-hydroxyl group is phosphoramidited” means that the oligonucleotide3′-hydroxyl group is modified by, for example, a phosphoramiditing grouprepresented by —P(OP²)(NR_(e)R_(f)) wherein each symbol is as definedabove.

The definitions, examples and preferable embodiments of P², R_(e) andR_(f) are as explained for the above-mentioned formula (I).

The definitions, examples and preferable embodiments of the “temporaryprotecting group removable under acidic conditions” are as explained forthe above-mentioned formula (I).

The definitions, examples and preferable embodiments of the “grouphaving a C₅₋₃₀ straight chain or branched chain alkyl group and/or aC₅₋₃₀ straight chain or branched chain alkenyl group” are as explainedfor the above-mentioned formula (I).

As the p-mer oligonucleotide comprising a protected base used in step(2), an oligonucleotide comprising a protected base represented by theabove-mentioned formula (I) is preferable.

4-8. Explanation of Steps (1)-(5)

While steps (1)-(5) are explained below by reference to the formulas(i), (ii), (iii) and the like for convenience, they are not limitedthereby.

Step (1) (Deprotection Step)

In this step, before condensation step (2), in a non-polar solvent,temporary protecting group P¹ (P¹ is a temporary protecting groupremovable under acidic conditions) of the 5′-terminal hydroxyl group ofan n-mer oligonucleotide (i) wherein the 3′-hydroxyl group is protected,and the 5′-hydroxyl group is protected by a temporary protecting groupremovable under acidic conditions is removed by reaction with an acid(deprotection step).

wherein each symbol is as defined above.

This step is performed in a solvent that does not influence thereaction. Since a higher solubility of the solvent is expected to affordsuperior reactivity, a non-polar solvent showing high solubility of then-mer oligonucleotide of the present invention is preferably selected.Specifically, examples thereof include halogenated solvents such aschloroform, dichloromethane, 1,2-dichloroethane and the like; aromaticsolvents such as benzene, toluene, xylene, mesitylene and the like;ester solvents such as ethyl acetate, isopropyl acetate and the like;aliphatic solvents such as hexane, pentane, heptane, octane, nonane,cyclohexane and the like; non-polar ether solvents such as diethylether, cyclopentyl methyl ether, tert-butyl methyl ether and the like.Two or more kinds of these solvents may be used in a mixture in anappropriate ratio. In addition, the above-mentioned non-polar solventmay be mixed with a polar solvent at an appropriate ratio, such asnitrile solvents such as acetonitrile, propionitrile and the like, amidesolvents such as N,N-dimethylformamide, N,N-dimethylacetamide,N-methylpiperidone and the like, as long as n-mer oligonucleotide isdissolved. Of these, aromatic solvents, aliphatic solvents, or acombination of these is preferable, benzene, toluene, hexane, pentane,heptane, nonane, cyclohexane or a combination of these is preferable,toluene, heptane, nonane or a combination of these is more preferable,and toluene and heptane or a combination of these is particularlypreferable.

In this step, the concentration of n-mer oligonucleotide (i) in asolvent is not particularly limited as long as the oligonucleotide isdissolved, it is preferably 1 to 30 mass %.

To continuously perform the deprotection step, subsequent condensationstep, and oxidation step in a solution, it is preferable to use a cationscavenger in this step during or after the removal reaction of atemporary protecting group P¹ of 5′-hydroxyl group in n-meroligonucleotide (i).

While the cation scavenger is not particularly limited as long asre-protection (returning to starting material) with the removedprotecting group P¹ and side reaction with the deprotected functionalgroup do not proceed, pyrrole derivatives such as pyrrole,2-methylpyrrole, 3-methylpyrrole, 2,3-dimethylpyrrole,2,4-dimethylpyrrole and the like; and indole derivatives such as indole,4-methylindole, 5-methylindole, 6-methylindole, 7-methylindole,5,6-dimethylindole, 6,7-dimethylindole and the like can be used. Since agood cation trap effect can be obtained, pyrrole, 3-methylpyrrole,2,4-dimethylpyrrole, indole, 4-methylindole, 5-methylindole,6-methylindole, 7-methylindole, 5,6-dimethylindole and6,7-dimethylindole are preferable, pyrrole, 3-methylpyrrole and indoleare more preferable, pyrrole and indole are more preferable, and pyrroleis particularly preferable.

The amount of cation scavenger to be used in this step is 1 to 50 mol,preferably 5 to 20 mol, per 1 mol of n-mer oligonucleotide (i).

While the acid to be used in this step is not particularly limited aslong as good deprotection can be achieved, trifluoroacetic acid,dichloroacetic acid, trifluoromethanesulfonic acid, trichloroaceticacid, methanesulfonic acid, hydrochloric acid, acetic acid,p-toluenesulfonic acid and the like are preferably used.

Since good reaction can be achieved, trifluoroacetic acid,dichloroacetic acid, trifluoromethanesulfonic acid and trichloroaceticacid are more preferable, trifluoroacetic acid, dichloroacetic acid andtrifluoromethanesulfonic acid are more preferable, trifluoroacetic acidand dichloroacetic acid are still more preferable, and trifluoroaceticacid is particularly preferable. These acids may be diluted with theabove-mentioned non-polar solvent. When the aforementioned acid is used,it may be combined with a particular base to appropriately adjust theacidity before use.

The amount of the acid to be used in this step is 1 to 100 mol,preferably 1 to 40 mol, per 1 mol of n-mer oligonucleotide (i).

While the reaction temperature in this step is not particularly limitedas long as the reaction proceeds, it is preferably −10° C. to 50° C.,more preferably 0° C. to 40° C. While the reaction time varies dependingon the kind of n-mer oligonucleotide to be used, the kind of acid, thekind of solvent, the reaction temperature and the like, it is 5 min to 5hr.

To continuously perform the deprotection step, subsequent condensationstep, and oxidation step in a solution, it is preferable to remove thetemporary protecting group of the 5′-hydroxy group in this step andneutralize the compound with an organic base.

The organic base to be used for neutralization is not particularlylimited as long as it can neutralize the above-mentioned acids, and theobtained salt can function as a condensing agent. Since the reactionproceeds smoothly, pyridine, benzimidazole, 1,2,4-triazole,N-phenylimidazole, 2-amino-4,6-dimethylpyrimidine, 1,10-phenanthroline,imidazole, N-methylimidazole, 2-chlorobenzimidazole,2-bromobenzimidazole, 2-methylimidazole, 2-phenylbenzimidazole,N-phenylbenzimidazole and 5-nitrobenzimidazole are preferable, pyridine,benzimidazole, 1,2,4-triazole, N-phenylimidazole, N-methylimidazole,2-amino-4,6-dimethylpyrimidine and 1,10-phenanthroline are morepreferable, pyridine, benzimidazole, 1,2,4-triazole andN-phenylimidazole are further preferable, pyridine, benzimidazole and1,2,4-triazole are still more preferable, and pyridine is particularlypreferable.

The amount of the organic base to be used in this step is 1 to 10 mol,preferably 1 to 3 mol, per 1 mol of acid.

A particularly preferable combination of an acid and an organic base inthis step is that of trifluoroacetic acid and pyridine and/orN-methylimidazole.

Step (2) (Condensation Step)

In this step, an n-mer oligonucleotide (ii) wherein the 5′-hydroxylgroup is not protected, and the 3′-hydroxyl group is protected iscondensed with a p-mer oligonucleotide comprising a protected base (iii)wherein the 3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl groupis protected by a temporary protecting group removable under acidicconditions, and the nucleic acid base is protected by a group having aC₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀straight chain or branched chain alkenyl group.

-   wherein X′ means the same as X, and other symbols are as defined    above.

In this step, the n-mer oligonucleotide (ii) is not particularlylimited, and one obtained in the aforementioned step (1) can bepreferably used. In this case, a p-mer oligonucleotide comprising aprotected base (iii) only needs to to be added directly to the reactionmixture after step (1), without isolating the n-mer oligonucleotide(ii). In this condensation step, the salt (e.g., pyridinetrifluoroacetate), which is formed by the acid added during deprotectionstep (1) and the organic base added during neutralization reaction, actsas a condensing agent. Therefore, steps (1) and (2) continuouslyperformed in a solution provide advantages of not only omission of anisolation operation but also improved reaction efficiency.

The reaction efficiency can also be improved by adding a condensingagent (e.g., pyridine trifluoroacetate, tetrazole,5-benzylthio-1H-tetrazole, 4,5-dicyanoimidazole etc.) in thiscondensation reaction.

In this step, moreover, when the acidity of the reaction mixture becomeshigh, a side reaction removing temporary protecting group P¹ may occur.Therefore, N-methylimidazole is preferably added to suppressacidification of the reaction mixture.

The amount of N-methylimidazole to be added to adjust the acidity is 0.1to 1 mol, preferably 0.5 mol, per 1 mol of organic base used forneutralization.

As the p-mer oligonucleotide comprising a protected base (iii) used inthis step, an oligonucleotide comprising a protected base represented bythe above-mentioned formula (I) can be preferably used.

This step is performed in a solvent that does not influence thereaction. Specifically, a non-polar solvent similar to the one used inthe aforementioned step (1) can be mentioned. For efficient activationof a phosphoramidite group of the p-mer oligonucleotide comprising aprotected base (iii), a mixture of the above-mentioned non-polar solventand a polar solvent, for example, nitrile solvents such as acetonitrile,propionitrile and the like; ketone solvents such as acetone, 2-butanoneand the like; N,N-dimethylformamide, N,N-dimethylacetamide, polar ethersolvents such as 1,4-dioxane, tetrahydrofuran and the like, amidesolvents such as N-methylpyrrolidone and the like; sulfoxide solventssuch as dimethyl sulfoxide and the like, and the like at an appropriateratio is preferably used as long as the n-mer oligonucleotide (ii) afterremoval of the temporary protecting group of the 5′-hydroxyl group canbe dissolved.

In this case, as the polar solvent, amide solvent, nitrile solvent, anda combination thereof are preferable, acetonitrile,N,N-dimethylformamide, N-methylpiperidone, and a combination thereof aremore preferable, and acetonitrile is particularly preferable.

The polar solvent may be added as a solution of a p-mer oligonucleotidecomprising a protected base (iii), a condensing agent and the like.

The amount of a p-mer oligonucleotide comprising a protected base (iii)to be used is 1 to 10 mol, preferably 1 to 5 mol, per 1 mol of an n-meroligonucleotide (ii).

While the reaction temperature is not particularly limited as long asthe reaction proceeds, 0° C. to 100° C. is preferable, and 20° C. to 50°C. is more preferable. While the reaction time varies depending on thekind of n-mer oligonucleotide to be condensed, the reaction temperatureand the like, it is 30 min to 24 hr.

Step (3) (Oxidation Step or Sulfurization Step)

The n+p-mer oligonucleotide (iv) obtained in step (2) is reacted with anoxidizing agent or sulfurizing agent to convert the phosphite triesterbond in the n+p-mer oligonucleotide (iv) to a phosphate triester bond ora thiophosphate triester bond.

wherein the symbols are as defined above.

This step can be simply performed by directly adding an oxidizing agentor sulfurizing agent to the reaction mixture after step (2), withoutisolating the n+p-mer oligonucleotide (iv) obtained in step (2).

While the “oxidizing agent” to be used in this step is not particularlylimited as long as it can oxidize a phosphite triester bond into aphosphate triester bond without oxidizing other moieties, iodine,(1S)-(+)-(10-camphorsulfonyl)oxaziridine, tert-butyl hydroperoxide(TBHP), 2-butanone peroxide, 1,1-dihydroperoxycyclododecane,bis(trimethylsilyl)peroxide, m-chloroperbenzoic acid or hydrogenperoxide is preferably used. Since good oxidation reaction can beachieved, iodine, (1S)-(+)-(10-camphorsulfonyl)oxaziridine, tert-butylhydroperoxide, 2-butanone peroxide and 1,1-dihydroperoxycyclododecaneare more preferable, iodine, (1S)-(+)-(10-camphorsulfonyl)oxaziridine,tert-butyl hydroperoxide and 2-butanone peroxide are more preferable,iodine and tert-butyl hydroperoxide are still more preferable, andiodine is particularly preferable. The oxidizing agent can be used afterdiluting with a suitable solvent to achieve a concentration of 0.05 to2M. While the dilution solvent is not particularly limited as long as itis inert to the reaction, for example, pyridine, THF, dichloromethane,water, nonane and a mixed solvent of any of them can be mentioned. Ofthese, for example, iodine/water/pyridine-THF, iodine/pyridine-aceticacid, peroxide (TBHP)/dichloromethane, tert-butyl hydroperoxide/nonaneor hydrogen peroxide/potassium iodide/phosphoric acid buffer arepreferably used.

The “sulfurizing agent” to be used in this step is not particularlylimited as long as it can convert a phosphite triester bond to athiophosphate triester bond,3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent),3H-1,2-benzodithiol-3-one, phenylacetyl disulfide (PADS),tetraethylthiuram disulfide (TETD), 3-amino-1,2,4-dithiazole-5-thione(ADTT) or sulfur is preferably used. Since a good reaction proceeds,3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione(DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent),3H-1,2-benzodithiol-3-one and phenylacetyl disulfide (PADS) are morepreferable,3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione and3H-1,2-benzodithiol-3-one-1,1-dioxide are further preferable, and3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione isparticularly preferable. The sulfurizing agent can be used afterdiluting with a suitable solvent to achieve a concentration of 0.05 to2M. While the dilution solvent is not particularly limited as long as itis inert to the reaction, for example, dichloromethane, acetonitrile,pyridine and a mixed solvent of any of them can be mentioned.

The amount of the oxidizing agent or sulfurizing agent to be used is 1to 50 mol, preferably 1 to 5 mol, per 1 mol of the n+p-meroligonucleotide (iv) obtained in step (2).

While the reaction temperature is not particularly limited as long asthe reaction proceeds, 0° C. to 100° C. is preferable, and 20° C. to 50°C. is more preferable. While the reaction time varies depending on thekind of n+p-mer oligonucleotide (iv), the kind of oxidizing agent orsulfurizing agent to be used, the reaction temperature and the like, itis 1 min to 3 hr.

Step (4) (Extraction Isolation Step)

This step is a method of isolating and purifying an n+p-meroligonucleotide (v) from a reaction mixture containing n+p-meroligonucleotide (v) having a phosphate triester bond or a thiophosphatetriester bond, which is obtained from step (3), by an extractionoperation alone.

While the extraction operation is not particularly limited, it ispreferably performed by adding a polar solvent and/or a non-polarsolvent as necessary to the reaction mixture obtained in step (3),partitioning the mixture between polar solvent-non-polar solvent, andtransferring the n+p-mer oligonucleotide to the non-polar solvent. Whenthe reaction is performed by mixing a non-polar solvent with a polarsolvent in step (2), the reaction mixture is preferably partitioned byadding a non-polar solvent.

The extraction operation can remove impurities such as the remainingstarting materials, reagents (e.g., acid, cation scavenger, organicbase, p-mer oligonucleotide wherein 3′-hydroxyl group isphosphoramidited and the 5′-hydroxyl group is protected by a temporaryprotecting group removable under acidic conditions, oxidant andsulfurizing agent) and the like into a polar solvent.

Examples of the non-polar solvent to be added as necessary to transferan n+p-mer oligonucleotide into a non-polar solvent in this step includehalogenated solvents such as chloroform, dichloromethane,1,2-dichloroethane and the like; aromatic solvents such as benzene,toluene, xylene, mesitylene and the like; ester solvents such as ethylacetate, isopropyl acetate and the like; aliphatic solvents such ashexane, pentane, heptane, octane, nonane, cyclohexane and the like;non-polar ether solvents such as diethyl ether, cyclopentyl methylether, tert-butyl methyl ether and the like. Two or more kinds of thesesolvents may be used in a mixture in an appropriate ratio. Of these,aromatic solvents, aliphatic solvents, or a combination of these ispreferable, benzene, toluene, hexane, pentane, heptane, nonane,cyclohexane or a combination of these is preferable, toluene, heptane,nonane or a combination of these is more preferable, toluene, heptane ora combination of these is further preferable, and heptane isparticularly preferable.

Examples of the polar solvent to be added as necessary to transferimpurities in this step into a polar solvent include alcohol solventmethanol, ethanol, isopropanol and the like, nitrile solvents such asacetonitrile, propionitrile and the like, ketone solvents such asacetone, 2-butanone and the like, polar ether solvents such as1,4-dioxane, tetrahydrofuran and the like, amide solvents such asN,N-dimethylformamide, N,N-dimethylacetamide, N-methylpiperidone and thelike, sulfoxide solvents such as dimethyl sulfoxide and the like, waterand the like, and a mixed solvent of two or more kinds of these. Ofthese, amide solvents, nitrile solvents, and a combination of these arepreferable, acetonitrile, N,N-dimethylformamide, N-methylpiperidone, anda combination of these are more preferably used. The polar solvent inthe present invention is particularly preferably acetonitrile from thepractical aspects.

The impurity can be removed by removing polar solvents afterpartitioning between polar solvent-non-polar solvent.

Moreover, the impurity remaining in a small amount can be furtherremoved by adding a polar solvent to a non-polar solvent after removalof the polar solvent, stirring the mixture and removing the polarsolvent by partitioning (washing in the present invention).

While the number of washing with a polar solvent is not particularlylimited, it may be repeated until the impurity in the non-polar solventlayer decreases to the extent the nucleotide elongation reaction in thenext cycle is not inhibited by the analysis of the non-polar solvent bythin layer silica gel chromatography, high performance liquidchromatography and the like.

The polar solvent to be used for partitioning and washing may containwater to improve partitioning performance from the non-polar solvent.

In this case, the water content of the polar solvent is preferably 1-10%(v/v), more preferably 3-8% (v/v). When the water content is too low,the partitioning performance may be insufficient, and when the watercontent is too high, the solubility of the byproduct, the remainingstarting material, reagent and the like to be removed in a polar solventtrends to decrease to degrade the removal efficiency.

The n+p-mer oligonucleotide (v) can be isolated by concentrating thenon-polar solvent layer after washing with a polar solvent. In thiscase, nucleotide elongation can be repeated in one-pot by adding thesolvent and reagent for the next cycle to a reaction vessel containingthe concentrate.

Alternatively, it is also possible to apply, without concentration, thenon-polar solvent layer after washing to the nucleotide elongation inthe next cycle.

The production method of oligonucleotide of the present invention canafford the object long oligonucleotide with high purity and in a highyield by repeating the above-mentioned steps (1) to (4) a desired numberof times.

Step (5) (Deprotection, Oligonucleotide Isolation Step)

In the production method of oligonucleotide of the present invention,deprotection is performed after step (4) according to the kind andproperties of the protecting group, whereby oligonucleotide is isolated.All protecting groups can be removed from oligonucleotide according tothe deprotection method described in Greene's PROTECTIVE GROUPS INORGANIC SYNTHESIS, 4th ed., JOHN WILLY&SONS (2006) and the like. To bespecific, nucleotide 3′-hydroxyl-protecting group, as well asphenoxyacetyl group, acetyl group, a group having a C₅₋₃₀ straight chainor branched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group and the like, which are nucleic acid base-protectinggroups, cyanoethyl group bonded to phosphate group and the like in thepresent invention can all be removed by treating with aqueousammonia/ethanol solution, aqueous ammonia/aqueous methylamine solution,ethylenediamine and the like. In addition, nucleotide 5′hydroxyl-protecting group can be removed by a treatment with the acidused in step (1) or an appropriately diluted solution of such acid.

Since oligonucleotide without a protecting group is easily degraded byan enzyme, oligonucleotide is preferably isolated under appropriate aircontamination control.

The progress of the reaction in each of the above-mentioned steps can beconfirmed by a method similar to conventional liquid phase organicsynthesis reaction. That is, the reaction can be traced by thin layersilica gel chromatography, high performance liquid chromatography andthe like.

The oligonucleotide obtained by step (4) or step (5) can also be led toa desired oligonucleotide derivative by further applying an organicsynthesis reaction.

5. Explanation of Steps (1′)-(5′)

Another embodiment of the production method of oligonucleotide of thepresent invention is a method including the following step (2′).

-   (2′) A step of producing an n′+p′-mer oligonucleotide by condensing    a p′-mer oligonucleotide (p′ is an integer of one or more) wherein    the 3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group is    protected by a temporary protecting group removable under acidic    conditions, and the nucleic acid base is optionally protected, with    an n′-mer oligonucleotide (n′ is an integer of one or more) wherein    the 5′-hydroxyl group is not protected, and the 3′-hydroxyl group is    protected by a protecting group represented by the above-mentioned    formula (III) to form a phosphite triester bond via the 5′-hydroxyl    group thereof.

This method preferably further includes the following step (3′), whereinthe phosphite triester bond of the n′+p′-mer oligonucleotide obtained instep (2′) is converted to a phosphate triester bond or a thiophosphatetriester bond: (3′) a step of adding an oxidizing agent or a sulfurizingagent to the reaction mixture obtained in the condensation step (2′) toconvert the phosphite triester bond of the n′+p′-mer oligonucleotideobtained in the condensation step to a phosphate triester bond or athiophosphate triester bond.

This method preferably further includes the following step (1′) by whichan n′-mer oligonucleotide used in step (2′) wherein the 5′-hydroxylgroup is not protected and the 3′-hydroxyl group is protected by aprotecting group represented by the above-mentioned formula (III) isprepared.

-   (1′) a step of removing a temporary protecting group removable under    acidic conditions of the 5′-hydroxyl group by reacting, in a    non-polar solvent prior to the condensation step (2′), the n′-mer    oligonucleotide wherein the 3′-hydroxyl group is protected by the    protecting group represented by the above-mentioned formula (III),    and the 5′-hydroxyl group is protected by the temporary protecting    group, with an acid.

Step (1′) is preferably performed in the presence of at least one kindof cation scavenger selected from a pyrrole derivative and an indolederivative, and further includes a step of neutralization with anorganic base after removal of the temporary protecting group of the5′-hydroxyl group. As a result, steps (1′), (2′) and (3′) can becontinuously performed in a liquid, and an oligonucleotide whereinnucleoside in the number of p′ is elongated can be isolated and purifiedby an extraction operation alone.

Furthermore, by including the following step (4′), an n′+p′-meroligonucleotide is purified by removing an excess starting material andby-product conveniently and effectively, without the need forcomplicated solidification-isolation, and can be led to the next stepwithout taking out the resultant product from the reaction vessel:

-   (4′) a step of isolating the n′+p′-mer oligonucleotide from the    reaction mixture obtained in step (3′) by an extraction operation    alone.

When the amount of the by-product generated can be controlled by themanagement of equivalent of the starting materials and controlling thereaction, it is preferable to repeat step (1′) to step (3′) as a basicunit, which includes step (4′).

Since the generation of by-product can be strictly managed andcontrolled and highly pure oligonucleotide can be obtained, it ispreferable to repeat step (1′) to step (4′) as a basic unit.

By repeating such cycle in the liquid phase method, the finaloligonucleotide can be produced in one-pot, without changing thereaction vessel.

In the production method of the present invention, oligonucleotide canbe isolated and produced by further including step (5′):

-   (5′) a step of removing all the protecting groups of the n′+p′-mer    oligonucleotide obtained in step (4).

n′ is an integer of one or more. While the upper limit of n is notparticularly limited, it is generally 100 or less, preferably 75 orless, more preferably 50 or less, and more preferably 30 or less

p′ is an integer of one or more, preferably 1. While the upper limit ofp is not particularly limited, it is preferably 50 or less, morepreferably 30 or less, more preferably 20 or less, still more preferably5 or less, and particularly preferably 3 or less.

The n′-mer oligonucleotide used in step (1′) is, for example, an n′-meroligonucleotide represented the following formula (i′) wherein P¹ is atemporary protecting group removable under acidic conditions, the3′-hydroxyl group is protected by a protecting group represented by theabove-mentioned formula (III) and the 5′-hydroxyl group is protected bya temporary protecting group removable under acidic conditions, and then′-mer oligonucleotide used in step (2′) is, for example, an n′-meroligonucleotide represented by the following formula (ii′) wherein the5′-hydroxyl group is not protected, and the 3′-hydroxyl group isprotected by a protecting group represented by the above-mentionedformula (III).

-   wherein m′ is any integer of not less than 0, and each of other    symbols is as defined above.

While the upper limit of m′ is not particularly limited, it is generally99 or less, preferably 74 or less, more preferably 49 or less, morepreferably 29 or less.

The definition, example and preferable embodiment of each of othersymbols are the same as those in the explanation on the formulas (i) and(ii).

The “a p′-mer oligonucleotide wherein the 3′-hydroxyl group isphosphoramidited, the 5′-hydroxyl group is protected by a temporaryprotecting group removable under acidic conditions, and the nucleic acidbase is optionally protected” used in step (2′) is not particularlylimited as long as the structural requirements are satisfied.

The “3′-hydroxyl group is phosphoramidited” means that oligonucleotide3′-hydroxyl group is modified by, for example, a phosphoramidite grouprepresented by —P(OP²)(NR_(e)R_(f)) wherein each symbol is as definedabove.

The definitions, examples and preferable embodiments of P², R_(e) andR_(f) are as explained for the above-mentioned formula (I).

The definitions, examples and preferable embodiments of “temporaryprotecting group removable under acidic conditions” are as explained forthe above-mentioned formula (I).

The protecting group of the “nucleic acid base is optionally protected”are the same as the protecting groups exemplified for the “optionallyprotected nucleic acid base” for Base in the above-mentioned formulas(i) and (ii).

The protecting group is preferably a group free of a C₅₋₃₀ straightchain or branched chain alkyl group and/or a C₅₋₃₀ straight chain orbranched chain alkenyl group and, for example, pivaloyl group,pivaloyloxymethyl group, trifluoroacetyl group, phenoxyacetyl group,4-isopropylphenoxyacetyl group, 4-tert-butylphenoxyacetyl group, acetylgroup, benzoyl group, isobutyryl group, dimethylformamidinyl group,9-fluorenylmethyloxycarbonyl group and the like are preferable. Ofthese, phenoxyacetyl group, 4-isopropylphenoxyacetyl group, acetylgroup, benzoyl group, isobutyryl group, and dimethylformamidinyl groupare more preferable.

Examples of the p′-mer oligonucleotide having optionally protectednucleic acid base wherein the 3′-hydroxyl group is phosphoramidited, andthe 5′-hydroxyl group is protected by a temporary protecting groupremovable under acidic conditions include the following formula (iii′).

wherein

-   Base is optionally substituted nucleic acid base, q′ is any integer    of not less than 0, and other symbol is as defined above.

“optionally substituted nucleic acid base” for Base is as defined forthe “optionally protected nucleic acid base” for Base in the formulas(i) and (ii).

q′ is any integer of 0 or more, preferably 0. While the upper limit ofq′ is not particularly limited, it is preferably 49 or less, morepreferably 29 or less, more preferably 19 or less, still more preferably4 or less, still more preferably 2 or less, and particularly preferably1.

Steps (1′)-(5′) can be performed under similar conditions as in theabove-mentioned steps (1)-(5) by reading the formulas (i), (ii) and(iii) as the formulas (i′), (ii′) and (iii′), respectively.

6. Use of Oligonucleotide

The oligonucleotide produced by the present invention can be used forvarious applications such as various veterinary pharmaceutical products(RNA, DNA, oligonucleic acid medicine, etc.) for human or animal,functional food, food for specified health uses, food, chemical product,polymer material for human or industrial use, and the like.

EXAMPLES

The present invention is explained in more detail in the following byreferring to Preparation Examples and Examples, which are not to beconstrued as limiting the scope of the present invention. The reagents,apparatuses and materials used in the present invention are commerciallyavailable unless otherwise specified. In the present specification, whenindicated by abbreviation, each indication is based on the abbreviationof the IUPAC-IUB Commission on Biochemical Nomenclature or conventionalabbreviations in the art.

The yield in the following Preparation Examples and Examples showsmol/mol %. Unless particularly specified, “%” means “mass %” in thepresent specification. In addition, the ratio of the solvent in thefollowing Preparation Examples and Examples is volume ratio. For ¹H-NMRspectrum, tetramethylsilane was used as the internal standard, and CDCl₃was used as a measurement solvent. NMR spectrum was measured usingBruker AVANCE AV300 (300 MHz) nuclear magnetic resonance apparatus orBruker AVANCE 400 (400 MHz) nuclear magnetic resonance apparatus.

For electrospray ionization liquid chromatography/mass spectrometry(hereinafter to be abbreviated as LC/MS), flow injection analysis (FIA)(solvent: 0.1 mol/l TEAA buffer pH 7.0, acetonitrile, ionization mode:ESI, ion node: negative, mass analyzer: quadrupole, fragmentor voltage:200V) was performed using 6130 Quadrupole LC/MS (Agilent Technologies).

For quadrupole mass spectrometry, flow injection analysis (FIA)(solvent: acetonitrile, ionization mode: ESI, ion mode:positive•negative, mass analyzer: quadrupole, fragmentor voltage: 71V)was performed using ZQ2000 (manufactured by Nihon Waters K.K.).

The abbreviations used in the following Preparation Examples andExamples are as described below. When nucleic acid base of nucleoside isprotected, the protecting group is shown in superscript after eachnucleoside.

-   dT: 2′-deoxythymidine-   dC: 2′-deoxycytidine-   dG: 2′-deoxyguanosine-   dA: 2′-deoxyadenosine-   U(M): 2′-methoxyuridine-   U(F): 2′-fluorouridine-   (LNA)T: 2′-O,4′-C-methylenethymidine-   DMTr: 4,4′-dimethoxytrityl-   PA: (2-cyanoethyl)-N,N-diisopropylphosphoramidite-   suc: succinyl-   TPB: 3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl (same    as 3,4,5-tris(2,3-phytyloxy)benzyl)-   Phy: 2,3-dihydrophytyl (wherein “2,3-dihydrophytyl” means    “3,7,11,15-tetramethyl-1-hexadecanyl”)-   PhyOM: (3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl (same as    2,3-phytyloxymethyl)-   4-Cit-Bz: 4-(3,7-dimethyl-1-octyloxy)benzoyl (same as    4-(dihydrocitronellyloxy)-benzyl)-   2Et-Hex: 2-ethyl-1-hexanoyl-   3,5,5-Me3Hex: 3,5,5-trimethyl-1-hexanoyl-   Me6Dodecanoyl: 2,2,4,8,10,10-hexamethyl-5-dodecanoyl-   2-HepUndecanoyl: 2-heptyl-1-undecanoyl-   2-HexDecanoyl: 2-hexyl-1-decanoyl-   Myr: tetradecanoyl (same as myristoyl)-   Me6Dodecanoyl: 2,2,4,8,10,10-hexamethyl-5-dodecanoyl-   N,N-Cit2-methylene: bis(3,7-dimethyl-octyl)amino-methylene (same as    N,N-bis-dihydrocitronellyl-methylene)-   Bz: benzoyl-   ibu: isobutyryl

Preparation Example 1 Synthesis of 2,3-dihydrophytol

Phytol (10.00 g, 33.7 mmol) was dissolved in methanol, Pt/C (2%, 1.00 g)was suspended therein and the suspension was stirred overnight under ahydrogen atmosphere. After completion of the reaction, the suspensionwas filtered to remove Pt/C, and the filtrate was concentrated to give2,3-dihydrophytol. This was used for the next reaction withoutpurification.

Preparation Example 2 Synthesis of 2,3-dihydrophytyl bromide

2,3-Dihydrophytol (33.7 mmol) was suspended in 48% hydrobromic acid (100ml), concentrated sulfuric acid (0.17 ml) was added dropwise and themixture was stirred at 100° C. overnight. The reaction mixture wascooled to room temperature, extracted with hexane (200 ml), and washedtwice with 5% aqueous sodium hydrogen carbonate solution (70 ml) andonce with 20% brine (70 ml). The organic layer was dried over sodiumsulfate, and the solvent in the filtrate was evaporated. The obtainedresidue was purified by silica gel column chromatography (short column,hexane alone) to give 2,3-dihydrophytyl bromide (“2,3-dihydrophytylgroup” is sometimes to be referred to as “Phy” hereunder) (10.41 g, 28.8mmol, 85% vs. phytol).

Preparation Example 3 Synthesis of 3,7,11-trimethyldodecan-1-ol

Using Farnesol (3.00 g, 13.5 mmol) and in the same manner as inPreparation Example 1, 3,7,11-trimethyldodecan-1-ol was obtained. Thiswas used for the next reaction without purification.

Preparation Example 4 Synthesis of 1-bromo-3,7,11-trimethyldodecane

Using 3,7,11-trimethyldodecan-1-ol obtained in Preparation Example 3 andin the same manner as in Preparation Example 2,1-bromo-3,7,11-trimethyldodecane (2.98 g, 10.2 mmol, 76% vs. Farnesol)was obtained.

Preparation Example 5 Synthesis of1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)]-1-phenylmethanamine

To 2,3-dihydrophytyl bromide (1.02 g, 2.82 mmol) were added DMF (15 ml),2-chloro-5-hydroxybenzophenone (0.99 g, 4.23 mmol) and K₂CO₃ (0.78 g,5.64 mmol), and the mixture was stirred at 90° C. for 3 hr. The reactionmixture was cooled to room temperature, ethyl acetate (25 ml) and 1mol/l hydrochloric acid (25 ml) were added and the mixture was stirredto allow layer separation. The aqueous layer was separated anddiscarded. The organic layer was washed twice with purified water (25ml), and the organic layer was evaporated under reduced pressure to give2-chloro-5-(2,3-dihydrophytyloxy)benzophenone.

To the aforementioned 2-chloro-5-(2,3-dihydrophytyloxy)benzophenone wereadded chloroform (20 ml), methanol (2 ml) and sodium borohydride (440mg, 11.6 mmol), and the mixture was stirred at 50° C. overnight. Thereaction mixture was cooled to room temperature, 1 mol/l hydrochloricacid (15 ml) was added dropwise in an ice bath to decompose unreactedsodium borohydride. The aqueous layer was discarded, and the organiclayer was washed twice with purified water (10 ml). The organic layerwas evaporated under reduced pressure, and moisture was azeotropicallydistilled with acetonitrile to give2-chloro-5-(2,3-dihydrophytyloxy)benzhydrol.

To 2-chloro-5-(2,3-dihydrophytyloxy)benzhydrol were added chloroform (20ml), DMF (43 μl, 559 μmol) and thionyl chloride (1.03 ml, 14.1 mmol),and the mixture was stirred at 50° C. for 4 hr. The reaction mixture wascooled to room temperature. The solvent was evaporated under reducedpressure, and the remaining thionyl chloride was azeotropicallydistilled with toluene to give1-chloro-1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane.

To the aforementioned1-chloro-1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane wereadded DMF (15 ml) and sodium azide (786 mg, 12.1 mmol), and the mixturewas stirred at 80° C. overnight. The reaction mixture was cooled to roomtemperature, ethyl acetate (20 ml) and hexane (20 ml) were added, andthe mixture was washed once with purified water (30 ml), and twice withpurified water (15 ml). The organic layer was evaporated under reducedpressure to give1-azido-1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane.

To the aforementioned1-azido-1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane wereadded THF (20 ml), purified water (2 ml) and triphenylphosphine (813 mg,3.10 mmol), and the mixture was stirred at 50° C. for 2 hr. The reactionmixture was cooled to room temperature, THF was evaporated, andliquid-separated three times with heptane (30 ml)-50% aqueousacetonitrile solution (15 ml) and the heptane layer was evaporated underreduced pressure. The residue was purified by silica gel columnchromatography (hexane:ethyl acetate=100:0→5:1) to give1-[(2-chloro-5-(2′,3′-dihydrophytyloxy)phenyl)]-1-phenylmethanamine asan oil (1.35 g, 2.63 mmol, yield 93% vs. 2,3-dihydrophytyl bromide).

Preparation Example 6 Synthesis of4,4′-bis(2,3-dihydrophytyloxy)benzhydryl alcohol

To 2,3-dihydrophytyl bromide (14.3 g, 39.6 mmol) were added DMF (120ml), 4,4′-dihydroxybenzophenone (4.04 g, 18.9 mmol) and potassiumcarbonate (7.82 g, 56.6 mmol), and the mixture was stirred at 80° C. for5 hr. The reaction mixture was cooled to room temperature, ethyl acetate(300 ml) and 1 mol/l hydrochloric acid (100 ml) were added and themixture was stirred to allow layer separation. The aqueous layer wasseparated and discarded. The organic layer was washed twice withpurified water (100 ml), and the organic layer was evaporated underreduced pressure to give 4,4′-bis(2,3-dihydrophytyloxy)benzophenone oil.This was dissolved in chloroform (60 ml) and methanol (10 ml), sodiumborohydride (4.49 g, 119 mmol) was added, and the mixture was stirred at60° C. for 3 hr. To the reaction mixture was added 1 mol/l hydrochloricacid (80 ml), and the mixture was concentrated. Ethyl acetate (100 ml)was added, and the mixture was washed successively with 1 mol/lhydrochloric acid and water. The organic layer was concentrated to give4,4′-bis(2,3-dihydrophytyloxy)benzhydryl alcohol oil.

Preparation Example 7 Synthesis of 3,4,5-tri(2,3-dihydrophytyloxy)benzylalcohol

2,3-Dihydrophytyl bromide (40.6 g, 112 mmol), methyl gallate (5.90 g,32.0 mmol) and potassium carbonate (22.14 g, 160 mmol) were suspended inDMF (400 ml), and the mixture was stirred at 110° C. overnight. Thereaction mixture was extracted with hexane (800 ml), washed with 1 mol/lhydrochloric acid (400 ml), 5% aqueous sodium hydrogen carbonatesolution (400 ml) and 20% brine (400 ml), dried over sodium sulfate andthe solvent in the filtrate was evaporated to give methyl3,4,5-tri(2,3-dihydrophytyloxy)benzoate (29.3 g, yield 93%).

The aforementioned methyl 3,4,5-tri(2,3-dihydrophytyloxy)benzoate (29.3g, 30.0 mmol) was dissolved in THF (400 ml), and diisobutylaluminumhydride (DIBAL) (1.0 mol/l toluene solution, 96 ml, 96 mmol) was addeddropwise over 30 min under a nitrogen atmosphere at 0° C. After stirringat room temperature overnight, 0.2 mol/l hydrochloric acid (50 ml) wasadded dropwise at 0° C. to quench the reaction. The solvent wasevaporated to about half, and the residue was dissolved in ethyl acetate(600 ml). The mixture was washed three times with 1 mol/l hydrochloricacid (300 ml), once with 5% aqueous sodium hydrogen carbonate solution(300 ml), and once with 20% brine (300 ml), and dried over sodiumsulfate. The solvent in the filtrate was evaporated to give3,4,5-tri(2,3-dihydrophytyloxy)benzyl alcohol (26.8 g, yield 94%).

Preparation Example 8 Synthesis of 3,4,5-tri(2,3-dihydrophytyloxy)benzylamine

3,4,5-Tri(2,3-dihydrophytyloxy)benzyl chloride (6.46 g, 6.63 mmol) wasdissolved in DMF-chloroform (60+20 ml), sodium azide (861 mg, 13.2 mmol)was added and the mixture was stirred at 70° C. for 2 hr. The reactionmixture was cooled to room temperature, ethyl acetate (160 ml) wasadded, and the mixture was washed twice with water (80 ml) and threetimes with 20% brine (50 ml), and dried over sodium sulfate. The solventin the filtrate was evaporated to give3,4,5-tri(2,3-dihydrophytyloxy)benzyl azide oil, which was directly usedfor the next step.

The aforementioned 3,4,5-tri(2,3-dihydrophytyloxy)benzyl azide oil wasdissolved in THF (80 ml), water (1.19 ml, 66.1 mmol) andtriphenylphosphine (1.91 g, 7.28 mmol) were added and the mixture wasstirred at 70° C. for 1 hr. After cooling to room temperature, thesolvent was evaporated, and the residue was dissolved in heptane (160ml). The mixture was washed three times with 50% aqueous acetonitrilesolution (50 ml) and twice with 20% brine (50 ml), and dried over sodiumsulfate. The solvent in the filtrate was evaporated. The obtainedresidue was purified by silica gel column chromatography (hexane:ethylacetate=5:1→chloroform:methanol:aqueous ammonia=100:10:1) to give3,4,5-tri(2,3-dihydrophytyloxy)benzyl amine (5.31 g, 5.32 mmol, yield80% vs. chloride product).

Preparation Example 9 Synthesis of 3,5-bis(2,3-dihydrophytyloxy)benzylalcohol

2,3-Dihydrophytyl bromide (895 mg, 2.48 mmol), methyl3,5-dihydroxybenzoate (204 mg, 1.21 mmol), and potassium carbonate (513mg, 3.71 mmol) were suspended in DMF (10 ml), and the suspension wasstirred at 100° C. for 7 hr. The reaction mixture was extracted withethyl acetate (30 ml), and the extract was washed three times with 1mol/l hydrochloric acid (10 ml) and 20% brine (10 ml), and dried oversodium sulfate. The solvent in the filtrate was evaporated to givemethyl 3,5-bis(2,3-dihydrophytyloxy)benzoate (0.78 g, yield 92%).

The aforementioned methyl 3,5-bis(2,3-dihydrophytyloxy)benzoate (0.70 g,1.00 mmol) was dissolved in THF (10 ml), and lithium aluminum hydride(2.0 mol/l THF solution, 1.2 ml, 2.4 mmol) was added dropwise under anitrogen atmosphere at 0° C. After stirring at room temperature for 5hr, water was added dropwise at 0° C. to quench the reaction. Thesolution was dissolved in ethyl acetate (30 ml), and the mixture waswashed three times with 1 mol/l hydrochloric acid (10 ml), and once with20% brine (20 ml), and dried over sodium sulfate. The solvent in thefiltrate was evaporated. The obtained residue was purified by silica gelcolumn chromatography (hexane alone→hexane:ethyl acetate=5:1) to give3,5-bis(2,3-dihydrophytyloxy)benzyl alcohol (0.61 g, yield 90%).

Preparation Example 10 Synthesis of 4-(2,3-dihydrophytyloxy)benzylalcohol

2,3-Dihydrophytyl bromide (600 mg, 1.66 mmol), 4-hydroxybenzaldehyde(223 mg, 1.83 mmol) and potassium carbonate (344 mg, 2.49 mmol) weresuspended in DMF (6 ml), and the suspension was stirred at 60° C. for 3days. The reaction mixture was cooled to room temperature, and extractedwith ethyl acetate (30 ml). The extract was washed three times with 1mol/l hydrochloric acid (6 ml), three times with 5% aqueous sodiumhydrogen carbonate solution (6 ml), and once with 20% brine (6 ml), anddried over sodium sulfate. The solvent in the filtrate was evaporated.The obtained residue was purified by silica gel column chromatography(hexane:ethyl acetate=15:1→5:1) to give4-(2,3-dihydrophytyloxy)benzaldehyde (640 mg, yield 100% vs.2,3-dihydrophytyl bromide).

The aforementioned 4-(2,3-dihydrophytyloxy)benzaldehyde (640 mg, 1.66mmol) was dissolved in THF-methanol mixed solution (7+0.3 ml), sodiumborohydride (110 mg, 90%, 2.62 mmol) was added at 0° C., and the mixturewas stirred at room temperature for 30 min. The reaction mixture wascooled to 0° C., and the reaction was quenched with 1 mol/l hydrochloricacid. Ethyl acetate (30 ml) was added and the mixture was washed threetimes with 1 mol/l hydrochloric acid (5 ml), three times with 5% aqueoussodium hydrogen carbonate solution (5 ml), and once with 20% brine (5ml), and dried over sodium sulfate. The solvent in the filtrate wasevaporated to give 4-(2,3-dihydrophytyloxy)benzyl alcohol (619 mg, 1.53mmol, yield 92% vs. 2,3-dihydrophytyl bromide).

Preparation Example 11 Synthesis of 4-(2,3-dihydrophytyloxy)benzyl amine

4-(2,3-Dihydrophytyloxy)benzyl alcohol (619 mg, 1.53 mmol) was dissolvedin chloroform (6 ml), thionyl chloride (167 μl, 2.29 mmol) was added andthe mixture was stirred for 5 hr. After completion of the reaction, thesolvent was evaporated to give 4-(2,3-dihydrophytyloxy)-benzyl chlorideoil, which was directly used for the next step.

The aforementioned 4-(2,3-dihydrophytyloxy)benzyl chloride (1.53 mmol)was dissolved in DMF-CHCl₃ mixed solvent (6+3 ml), sodium azide (298 mg,4.58 mmol) was added and the mixture was stirred at 70° C. overnight.The reaction mixture was cooled to room temperature, ethyl acetate (20ml) was added, and the mixture was washed 5 times with water (10 ml) anddried over sodium sulfate. The solvent in the filtrate was evaporated togive 4-(2,3-dihydrophytyloxy)benzyl azide (632 mg, yield 96% vs.4-(2,3-dihydrophytyloxy)benzyl alcohol).

The aforementioned 4-(2,3-dihydrophytyloxy)benzyl azide (632 mg, 1.47mmol) was dissolved in THF (6 ml), water (265 μl, 14.7 mmol) andtriphenylphosphine (424 mg, 1.62 mmol) were added and the mixture wasstirred at 70° C. overnight. After cooling to room temperature, thesolvent was evaporated, and the residue was dissolved in hexane (10 ml),and the mixture was washed 3 times with 50% aqueous acetonitrilesolution (5 ml). The solvent was evaporated. The obtained residue waspurified by silica gel column chromatography (hexane:ethylacetate=5:1→chloroform:methanol:aqueous ammonia=50:5:1) to give4-(2,3-dihydrophytyloxy)benzyl amine (555 mg, 1.37 mmol, yield 94%).

Preparation Example 12 Synthesis of2-methoxy-4-(2,3-dihydrophytyloxy)benzyl amine

2,3-Dihydrophytyl bromide (2.00 g, 5.53 mmol),2-methoxy-4-hydroxybenzaldehyde (884 mg, 5.81 mmol) and potassiumcarbonate (1.15 g, 8.32 mmol) were suspended in DMF (20 ml), and thesuspension was stirred at 80° C. overnight. The reaction mixture wascooled to room temperature, and extracted with ethyl acetate (50 ml).The extract was washed three times with 1 mol/l hydrochloric acid (20ml), three times with 5% aqueous sodium hydrogen carbonate solution (20ml), and once with 20% brine (20 ml), and dried over sodium sulfate. Thesolvent in the filtrate was evaporated. The obtained residue waspurified by silica gel column chromatography (hexane:ethyl acetate=20:1)to give 2-methoxy-4-(2,3-dihydrophytyloxy)benzaldehyde oil, which wasused for the next step.

The aforementioned 2-methoxy-4-(2,3-dihydrophytyloxy)benzaldehyde, andhydroxylamine hydrochloride (1.15 g, 16.5 mmol) were suspended indichloromethane (25 ml), triethylamine (3.84 ml, 27.7 mmol) was added at0° C. and the suspension was stirred at room temperature for 3 hr. Tothe reaction mixture was added chloroform (30 ml) and the mixture waswashed three times with 1 mol/l hydrochloric acid (15 ml), three timeswith 5% aqueous sodium hydrogen carbonate solution (15 ml), and oncewith 20% brine (15 ml), and the solvent was evaporated to give2-methoxy-4-(2,3-dihydrophytyloxy)benzaldoxime. After confirmation ofthe structure by NMR, it was used for the next step.

The aforementioned 2-methoxy-4-(2,3-dihydrophytyloxy)benzaldoxime wasdissolved in methanol-THF mixed solvent (20+10 ml), 10%palladium-carbon(K) (200 mg) was added and the mixture was stirred undera hydrogen atmosphere at room temperature overnight. The solvent in thefiltrate was evaporated. The obtained residue was purified by silica gelcolumn chromatography (chloroform:methanol:aqueous ammonia=100:10:1) togive 2-methoxy-4-(2,3-dihydrophytyloxy)benzyl amine (1.87 g, 4.31 mmol,yield 78% vs.

2,3-dihydrophytyl bromide).

Preparation Example 13 Synthesis of4-(2,3-dihydrophytyloxy)-2-methylbenzyl alcohol

To methanol (10 ml) was added dropwise thionyl chloride (1.92 ml, 26.3mmol) at 0° C., 4-hydroxy-2-methylbenzoic acid (2.00 g, 13.1 mmol) wasadded, and the mixture was stirred at 60° C. overnight. After completionof the reaction, the solvent was evaporated, and the residue wasdissolved in ethyl acetate (20 ml). The mixture was washed twice with 5%aqueous sodium hydrogen carbonate solution (10 ml), once with 1 mol/lhydrochloric acid (10 ml), and once with water (10 ml), and the solventwas evaporated to give methyl 4-hydroxy-2-methylbenzoate (2.24 g, yield100%).

The aforementioned methyl 4-hydroxy-2-methylbenzoate (269 mg, 1.62mmol), 2,3-dihydrophytyl bromide (389 mg, 1.08 mmol) and potassiumcarbonate (297 mg, 2.15 mmol) were suspended in DMF (5 ml), and thesuspension was stirred at 90° C. for 5 hr. The reaction mixture wascooled to room temperature, extracted with hexane-ethyl acetate (10+10ml), and washed once with 1 mol/l hydrochloric acid (15 ml) and twicewith water (10 ml). The solvent was evaporated to give methyl4-(2,3-dihydrophytyloxy)-2-methylbenzoate. After confirmation of thestructure by NMR, it was used for the next step.

The aforementioned methyl 4-(2,3-dihydrophytyloxy)-2-methylbenzoate(1.08 mmol) was dissolved in THF (6 ml), DIBAL (1.0M, 4.9 ml, 4.9 mmol)was added, and the mixture was stirred at room temperature for 100 min.The reaction mixture was cooled to 0° C., and the reaction was quenchedwith 1 mol/l hydrochloric acid (15 ml). Hexane (10 ml) and ethyl acetate(10 ml) were added to allow liquid-separation, and the mixture waswashed once with 0.5 mol/l hydrochloric acid (10 ml) and once with water(10 ml), and the solvent was evaporated to give4-(2,3-dihydrophytyloxy)-2-methylbenzyl alcohol.

Preparation Example 14 Synthesis of4-(2,3-dihydrophytyloxy)-2-methylbenzyl amine

4-(2,3-Dihydrophytyloxy)-2-methyl-benzyl alcohol (1.08 mmol) wasdissolved in chloroform (8 ml), thionyl chloride (393 μl, 5.38 mmol) wasadded and the mixture was stirred at 50° C. for 4.5 hr. After completionof the reaction, the solvent was evaporated to give4-(2,3-dihydrophytyloxy)-2-methylbenzyl chloride. After confirmation ofthe structure by NMR, it was used for the next step.

The aforementioned 4-(2,3-dihydrophytyloxy)-2-methylbenzyl chloride(1.08 mmol) was dissolved in DMF (6 ml), sodium azide (350 mg, 5.38mmol) was added and the mixture was stirred at 70° C. overnight. Thereaction mixture was cooled to room temperature, hexane (10 ml) andethyl acetate (5 ml) were added, and the mixture was washed 3 times withwater (10 ml).

The solvent in the filtrate was evaporated to give4-(2,3-dihydrophytyloxy)-2-methylbenzyl azide. After confirmation of thestructure by NMR, it was used for the next step.

The aforementioned 4-(2,3-dihydrophytyloxy)-2-methylbenzyl azide (1.08mmol) was dissolved in THF (10 ml), water (2 ml) and triphenylphosphine(565 mg, 2.15 mmol) were added and the mixture was stirred at 60° C. for3 hr. After cooling to room temperature, the solvent was evaporated, andthe residue was dissolved in heptane (10 ml), and washed 3 times with50% aqueous acetonitrile solution (10 ml). The solvent was evaporatedand the obtained residue was purified by silica gel columnchromatography (hexane:ethyl acetate=5:1→chloroform:methanol:aqueousammonia=50:5:1) to give 4-(2,3-dihydrophytyloxy)-2-methylbenzyl amine(281 mg, 0.67 mmol, yield 62% vs. 2,3-dihydrophytyl bromide).

Preparation Example 15 Synthesis of2,2,4,8,10,10-hexamethyl-5-dodecanoic acid (4-hydroxymethyl)phenylamide

2,2,4,8,10,10-Hexamethyl-5-dodecanoic acid (2.81 g, 9.88 mmol),4-aminobenzyl alcohol (1.00 g, 8.12 mmol) and3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt) (133 mg, 0.812mmol) were suspended in chloroform (10 ml), EDC HCl (2.05 g, 10.7 mmol)was added at 0° C., and the suspension was stirred at room temperatureovernight. The solvent was removed, and the residue was purified bysilica gel column chromatography (hexane:ethyl acetate=2:1) to give2,2,4,8,10,10-hexamethyl-5-dodecanoic acid (4-hydroxymethyl)phenylamide(2.69 g, 6.67 mmol, yield 82%).

Preparation Example 16 Synthesis of 4-(3,7,11-trimethyldodecyloxy)benzylalcohol

1-Bromo-3,7,11-trimethyldodecane (1.00 g, 3.43 mmol) obtained inReference Example 4 was dissolved in DMF (5 ml), 4-hydroxybenzyl alcohol(0.85 g, 6.85 mmol) and potassium carbonate (1.42 g, 10.3 mmol) wereadded and the mixture was stirred at 120° C. overnight. The reactionmixture was cooled to room temperature, extracted with chloroform (50ml), and the extract was washed three times with 1 mol/l hydrochloricacid (30 ml), once with 5% aqueous sodium hydrogen carbonate solution(30 ml), and once with purified water (30 ml). The solvent of theorganic layer was evaporated to give4-(3,7,11-trimethyldodecyloxy)benzyl alcohol (1.09 g, 3.26 mmol, yield95% vs. 1-bromo-3,7,11-trimethyldodecane).

Preparation Example 17 Synthesis of 3,7,11,15-tetramethyl-1-hexadecanoicacid

3,7,11,15-Tetramethyl-hexadecan-1-ol (8.96 g, 30.0 mmol) was dissolvedin a mixed solvent of acetone (360 ml) and acetic acid (180 ml), asolution of anhydrous chromic acid (7.27 g, 72.7 mmol) in water (9.0 ml)was added dropwise, and the mixture was stirred at room temperature for1 hr. A solution of sodium disulfite (100 g, 526 mmol) in water (450 ml)was added to the reaction mixture after completion of the reaction, themixture was stirred at room temperature overnight and extracted 6 timeswith diethyl ether (135 ml). The solvent of the obtained organic layerwas evaporated under reduced pressure. Diethylether (450 ml) and water(150 ml) were added to the oil after concentration to allowpartitioning, and the aqueous layer was extracted 3 times with ethylacetate (150 ml). The combined organic layer was dried over anhydroussodium sulfate, and the solvent was evaporated under reduced pressure.The oil (10.1 g) after concentration was purified by chromatography(silica gel; 250 g, eluate; 50:1→3:1 hexane-ethyl acetate) to give thetitle compound (6.78 g, 72.3%) as a pale-blue oil.

Preparation Example 18 Synthesis of 3,7,11,15-tetramethyl-1-hexadecanoylchloride

The compound (2.81 g, 9.0 mmol) synthesized in Preparation Example 17was dissolved in anhydrous chloroform (4.5 ml), thionyl chloride (1.31ml, 18.0 mmol) was added and the mixture was stirred at room temperaturefor 1 hr. After completion of the reaction, the solvent was evaporatedunder reduced pressure and the obtained oil (3.07 g) was directly usedfor the next reaction as the title compound.

Preparation Example 19 Synthesis of chloromethyl3,7,11,15-tetramethyl-1-hexadecanoate

The compound (6.56 g, 21.0 mmol) synthesized in Preparation Example 17was dissolved in anhydrous chloroform (10 ml), thionyl chloride (3.06ml, 42.0 mmol) was added and the mixture was stirred at room temperaturefor 1 hr. After completion of the reaction, the solvent was evaporatedunder reduced pressure, and the obtained oil was added dropwise to a tomixed solid of 90% para-formaldehyde (840 mg, 25.2 mmol) and zincchloride (42.9 mg, 0.32 mmol) over 30 min under cooling in an ice bath.The obtained mixed solution was stirred with heating at 60° C. for 5 hrand allowed to cool to room temperature. 10% Aqueous sodium hydrogencarbonate solution (30 ml) and dichloromethane (15 ml) were added toallow layer separation, and the mixture was further extracted twice withan equal amount of dichloromethane. The combined organic layer waswashed with saturated brine (30 ml), dried over anhydrous sodium sulfateand the solvent was evaporated under reduced pressure. The oil (7.40 g)after concentration was purified by chromatography (silica gel; 120 g,eluate; 10:1 hexane-dichloromethane) to give the title compound (5.01 g,66.1%) as a colorless oil.

Preparation Example 20 Synthesis of 4-(3,7-dimethyl-1-octyloxy)benzoicacid (1) Synthesis of 1-bromo-3,7-dimethyloctane

3,7-Dimethyloctan-1-ol (10.0 g, 63.2 mmol) was suspended in 48% aqueoushydrobromic acid solution, concentrated sulfuric acid (0.17 ml) wasadded dropwise and the mixture was stirred at 100° C. for 16 hr. Thereaction mixture was allowed to cool to room temperature, extracted withhexane (200 ml), and washed twice with 5% aqueous sodium hydrogencarbonate solution (100 ml) and once with 20% brine (100 ml). Theorganic layer was dried over sodium sulfate, and the solvent in thefiltrate was evaporated to give the title compound (13.3 g, 95.1%) as acolorless oil.

(2) Synthesis of methyl [4-(3,7-dimethyl-1-octyloxy)]benzoate

Under an argon atmosphere, potassium carbonate (12.5 g, 90.3 mmol) wassuspended in anhydrous N,N-dimethylformamide (100 ml),1-bromo-3,7-dimethyloctane (13.3 g, 60.1 mmol) and methyl(4-hydroxy)benzoate (8.74 g, 57.4 mmol) were added, and the mixture wasstirred at 70° C. for 16 hr. The reaction mixture was filtered to removepotassium carbonate. Water (50 ml) was added and the mixture wasextracted with hexane (250 ml). The extract was washed successively with1.0 mol/l aqueous hydrochloric acid solution (100 ml), aqueous sodiumhydrogen carbonate solution (100 ml) and saturated brine (100 ml). Theobtained organic layer was dried over sodium sulfate, filtered andconcentrated under reduced pressure to give the title compound (16.1 g,95.7%) as a colorless oil.

(3) Synthesis of 4-(3,7-dimethyl-1-octyloxy)benzoic acid

Methyl[4-(3,7-dimethyl-1-octyloxy)]benzoate (16.1 g, 54.9 mmol) wasdissolved in 1,4-dioxane (300 ml), 50% aqueous potassium hydroxidesolution (25 ml) was added and the mixture was stirred at 100° C. for 6hr. To acidify the reaction mixture, concentrated hydrochloric acid wasadded dropwise, and the mixture was extracted with ethyl acetate (200ml), and washed with 10% aqueous sodium hydroxide solution (100 ml) andsaturated brine (100 ml). The organic layer was dried over sodiumsulfate, filtered and concentrated under reduced pressure to give thetitle compound (14.2 g, 92.8%) as a white solid.

Preparation Example 21 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-N⁴-acetyl-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](3.5 g, 4.5 mmol) was dissolved in 2.0 mol/l ammonia/methanol solution(40 ml) and the mixture was stirred at room temperature for 2 hr. Thereaction mixture was concentrated under reduced pressure to give thetitle compound (3.4 g) quantitatively.

Preparation Example 22 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-N²-(2-methyl-1-oxopropyl)-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](5.0 g, 5.9 mmol) was dissolved in 2.0 mol/l ammonia/methanol solution(60 ml) and the mixture was stirred at room temperature overnight. Thereaction mixture was concentrated under reduced pressure, and theobtained oil was purified by silica gel column chromatography to givethe title compound (4.6 g) quantitatively.

(Preparation of Amidine-Type Protecting Group Reagent)

Preparation Example 23 Synthesis of N,N-di(3,7-dimethyl-octyl)formamidedimethylacetal (1) Synthesis of 3,7-dimethyl-1-octyl bromide

48% HBr (200 ml) and concentrated sulfuric acid (0.46 ml) were added to3,7-dimethyl-1-octanol (21.0 g, 157.9 mmol), and the mixture was heatedovernight. After allowing to cool to room temperature, and the mixturewas extracted with hexane. The organic layer was washed with 5% aqueoussodium hydrogen carbonate solution and saturated brine, dried andconcentrated to give the title compound (28.7 g, 82.1%).

(2) Synthesis of di(3,7-dimethyl-octyl)benzyl amine

Benzyl amine (7.1 ml, 64.8 mmol), potassium carbonate (17.9 g, 129.6mmol) and the compound (28.7 g, 129.6 mmol) obtained in PreparationExample 23-(1) were dissolved in dry acetonitrile (80 ml) and themixture was heated overnight. The reaction mixture was concentrated,dichloromethane (200 ml) was added and the mixture was washed withwater. The organic layer was concentrated and the obtained oil waspurified by silica gel column chromatography to give the title compound(13.2 g, 52.2%).

(3) Synthesis of N,N-di(3,7-dimethyl-octyl)amine

The compound (13.2 g, 33.8 mmol) obtained in Preparation Example 23-(2)was dissolved in ethanol (150 ml), 5% palladium carbon (53% wetted, 2.86g) was added and the mixture was stirred overnight under a hydrogenatmosphere. The reaction mixture was filtered through celite to removethe palladium catalyst, and the filtrate was concentrated to give thetitle compound (10.0 g, 99.3%) quantitatively.

(4) Synthesis of N,N-di(3,7-dimethyl-octyl)formamide dimethylacetal

To the compound (7.0 g, 23.4 mmol) obtained in Preparation Example23-(3) were added N,N-dimethylformamide dimethyl acetal (2.8 g, 23.4mmol) and a catalytic amount of pyridinium p-toluenesulfonate, and themixture was heated to 160° C. and stirred overnight. The reactionmixture was evaporated under reduced pressure to give the title compound(1.4 g, 16%).

Preparation Example 24 Synthesis of2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl amine (1) Synthesis of2,3,4-tris(2,3-dihydrophytyloxy)benzophenone

Under an argon atmosphere, 2,3,4-trihydroxybenzophenone (0.94 g, 4.07mmol), 2,3-dihydrophytyl bromide (6.01 g, 16.6 mmol) and potassiumcarbonate (2.57 g, 138.2 mmol) were added to anhydrousN,N-dimethylformamide (25 ml), and the mixture was stirred at 80° C.overnight. The reaction mixture was allowed to cool to room temperature,extracted with hexane (50 ml), washed successively with 1 mol/l aqueoushydrochloric acid (20 ml), 5% aqueous sodium hydrogen carbonate solution(20 ml) and saturated brine (20 ml), and dried over sodium sulfate. Thesolvent in the filtrate was evaporated and the obtained residue waspurified by silica gel column chromatography (hexane:ethylacetate=100:0-95/5 (v/v)). The object fractions were collected andconcentrated to give the title compound (3.90 g, 88.9%) as an oil.

(2) Synthesis of 2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl alcohol

2,3,4-Tris(2,3-dihydrophytyloxy)benzophenone (3.90 g, 3.64 mmol)synthesized in Preparation Example 24-(1) was dissolved in a mixedsolvent of chloroform (35 ml) and methanol (3.5 ml), sodium borohydride(0.41 g, 10.9 mmol) was added, and the mixture was stirred at 45° C. for2 hr. After completion of the reaction, 0.1 mol/l aqueous hydrochloricacid was added dropwise to decompose unreacted sodium borohydride, andthe mixture was washed with 1.0 mol/l aqueous hydrochloric acid. Theorganic layer was dried over sodium sulfate and filtered, and thefiltrate was concentrated under reduced pressure. The solvent in thefiltrate was evaporated and the obtained residue was purified by silicagel column chromatography (hexane:ethyl acetate=98:2-90/10 (v/v)). Theobject fractions were collected and concentrated to give the titlecompound (3.56 g, 91.0%) as an oil.

(3) Synthesis ofN-(9-fluorenylmethoxycarbonyl)-2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylamine

Under an argon atmosphere, 2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylalcohol (3.56 g, 3.31 mmol) synthesized in Preparation Example 24-(2)and 9-fluorenylmethyl carbamate (1.42 g, 5.96 mmol) were dissolved inanhydrous toluene (40 ml) at 50° C., methanesulfonic acid (64 μl, 993μmol) was added and the mixture was stirred at 100° C. for 2 hr. Thereaction mixture was allowed to cool to room temperature, washed with 5%aqueous sodium hydrogen carbonate solution (20 ml) and saturated brine(20 ml), and dried over sodium sulfate. The solvent in the filtrate wasevaporated and the obtained residue was purified by silica gel columnchromatography (hexane:ethyl acetate=98:2-90/10 (v/v)). The objectfractions were collected and concentrated to give the title compound(4.00 g, 93.3%) as an oil.

(4) Synthesis of 2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl amine

N-(9-Fluorenylmethoxycarbonyl)-2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylamine (4.00 g, 3.09 mmol) synthesized in Preparation Example 24-(3) wasdissolved in a mixed solvent of chloroform (30 ml) and acetonitrile (15ml), 20% piperidine [1-methyl-2-pyrrolidone solution] (30.5 ml, 61.8mmol) was added, and the mixture was stirred at room temperature for 30min. After completion of the reaction, ethyl acetate (60 ml), hexane (30ml) and water (10 ml) were added to allow layer separation. The organiclayer was washed with saturated brine and dried over sodium sulfate. Thesolvent in the filtrate was evaporated and the obtained residue waspurified by silica gel column chromatography (hexane:ethylacetate=100:0-80/20 (v/v)). The object fractions were collected andconcentrated to give the title compound (2.92 g, 88.2%) as an oil.

Preparation Example 25 Synthesis of4,4′-bis(2,3-dihydrophytyloxy)benzhydryl amine (1) Synthesis ofN-(9-fluorenylmethoxycarbonyl)-bis-4-(2,3-dihydrophytyloxy)benzhydrylamine

To 4,4′-bis(2,3-dihydrophytyloxy)benzhydryl alcohol (3.80 g, 4.89 mmol)described in Preparation Example 6 were added toluene (50 ml) and9-fluorenylmethyl carbamate (2.11 g, 8.81 mmol), and the mixture wasdissolved by heating to 50° C. Methanesulfonic acid (95.3 μl, 1.47 mmol)was added and the mixture was stirred at 100° C. for 2 hr. Thecompletion of the reaction was confirmed and the reaction mixture wasallowed to cool to room temperature. 5% Aqueous sodium hydrogencarbonate solution (20 ml) was added and the mixture was stirred. Afterpartitioning, the organic layer was further washed with water (20 ml)and saturated brine (20 ml). The organic layer was evaporated underreduced pressure to give the title compound (5.10 g, quant).

(2) Synthesis of 4,4′-bis(2,3-dihydrophytyloxy)benzhydryl amine

N-(9-Fluorenylmethoxycarbonyl)-4-bis(2,3-dihydrophytyloxy)benzhydrylamine (5.10 g, 5.27 mmol) obtained in Preparation Example 25-(1) wasdissolved in a mixed solvent of chloroform (50 ml) and acetonitrile (25ml), 20% piperidine [1-methyl-2-pyrrolidone solution] (52.1 ml, 105.4mol) was added, and the mixture was stirred at room temperature for 30min. After completion of the reaction, ethyl acetate (100 ml), hexane(60 ml) and water (15 ml) were added to allow layer separation. Theorganic layer was washed with saturated brine and dried over sodiumsulfate. The solvent in the filtrate was evaporated and the obtainedresidue was purified by silica gel column chromatography (hexane:ethylacetate=100:0-30/70 v/v)). The object fractions were collected andconcentrated to give the title compound (3.56 g, 86.8%) as an oil.

Preparation Example 26 Synthesis of 3,5-bis(2,3-dihydrophytyloxy)benzylamine (1) Synthesis of 3,5-bis(2,3-dihydrophytyloxy)benzyl chloride

Under an argon atmosphere, 3,5-bis(2,3-dihydrophytyloxy)benzyl alcohol(4.70 g, 6.70 mmol) described in Preparation Example 9 was dissolved inchloroform (34 ml), pyridine (a few drops) and thionyl chloride (0.97ml, 13.4 mmol) were added, and the mixture was stirred at roomtemperature for 90 min. The reaction mixture was concentrated underreduced pressure to give the title compound (4.93 g, quant) as an oil.

(2) Synthesis of 3,5-bis(2,3-dihydrophytyloxy)benzyl azide

3,5-Bis(2,3-dihydrophytyloxy)benzyl chloride (4.93 g, 6.85 mmol)obtained in Preparation Example 26-(1) was dissolved in a mixed solventof chloroform (27 ml) and N,N-dimethylformamide (81 ml), sodium azide(0.90 g, 13.7 mmol) was added and the mixture was stirred at 80° C. for2.5 hr. After completion of the reaction, the reaction mixture wasallowed to cool to room temperature, ethyl acetate (250 ml) and purifiedwater (180 ml) were added to allow layer separation, and the organiclayer was washed with purified water (180 ml) and saturated brine (130ml). The organic layer was dried over sodium sulfate and filtered togive the title compound (4.79 g, 96.4%).

(3) Synthesis of 3,5-bis(2,3-dihydrophytyloxy)benzyl amine

Under an argon atmosphere, 3,5-bis(2,3-dihydrophytyloxy)benzyl azide(4.79 g, 6.60 mmol) obtained in Preparation Example 26-(2) was dissolvedin anhydrous tetrahydrofuran (33 ml), lithium aluminum hydride (0.50 g,13.2 mmol) was added under ice-cooling, and the reaction mixture wasstirred at room temperature for 2 hr. To the reaction mixture aftercompletion of the reaction were added dropwise 1 mol/l aqueoushydrochloric acid (25 ml) and ethyl acetate (50 ml) to allow layerseparation, and the aqueous layer was extracted with ethyl acetate (50ml). The organic layers were combined, washed twice with water (90 ml),and further washed with 5% aqueous sodium hydrogen carbonate solution(90 ml) and saturated brine (90 ml). The organic layer was dried overmagnesium sulfate, and the filtrate was concentrated under reducedpressure and purified by silica gel column chromatography to give thetitle compound (3.29 g, 71.1%) as an oil.

Preparation Example 27 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1) Synthesis of 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine

An operation of dissolving 2′-Deoxy-2′-fluorouridine (3.00 g, 12.2 mmol)in dry pyridine, followed by concentration under reduced pressure wasrepeated 3 times to perform dehydrative azeotropic distillation.Thereafter, under an argon atmosphere, the reaction mixture wasdissolved in dry pyridine (120 ml), 4,4′-dimethoxytrityl chloride (4.55g, 13.4 mmol) was added, and the mixture was stirred at room temperaturefor 3 hr. The completion of the reaction was confirmed, and ethylacetate (150 ml) and water (60 ml) were added to the reaction mixture toallow layer separation. The organic layer was washed 3 times with 5%aqueous sodium hydrogen carbonate solution (20 ml), washed with water(20 ml) and saturated brine (20 ml), and the obtained organic layer wasdried over sodium sulfate. The solvent in the filtrate was evaporatedand the obtained residue was purified by silica gel columnchromatography (hexane:ethyl acetate=50:50-0/100 (v/v), containing 1%triethylamine). The object fractions were collected and concentrated togive the title compound (8.48 g, quant).

(2) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-(4,4′-Dimethoxytrityl)-2′-deoxy-2′-fluorouridine (3.00 g, 5.47mmol) obtained in Preparation Example 27-(1) was dissolved in anhydrousdichloromethane (50 ml) under an argon atmosphere,N,N-diisopropylethylamine (0.55 ml, 3.18 mmol), 1H-tetrazole (0.45 g,6.45 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite(1.92 g, 6.36 mmol) were added, and the mixture was stirred at roomtemperature overnight. The completion of the reaction was confirmed, 5%aqueous sodium hydrogen carbonate solution (20 ml) was added to thereaction mixture to allow layer separation, and the organic layer waswashed with saturated brine (20 ml). The organic layer was dried oversodium sulfate, the filtrate was concentrated and the obtained crudeproduct was purified by silica gel chromatography (hexane:ethylacetate=75:25-30/70 (v/v), containing 3% triethylamine). The objectfractions were collected and concentrated to give the title compound(2.76 g, 67.3%).

Example 1 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-O-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinate[5′-O-DMTr-dT-suc-TPB] (1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate

Under an argon atmosphere, 5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine(4.98 g, 9.14 mmol) and tetrahydrofuran-2,5-dione (1.39 g, 13.9 mmol)were dissolved in anhydrous dichloromethane (100 ml), triethylamine(3.80 ml, 27.3 mmol) was added and the mixture was stirred at roomtemperature for 16 hr. The reaction mixture after completion of thereaction was washed 3 times with 2.0 mol/l aqueous triethylammoniumphosphate solution (70 ml), and the organic layer was dried over sodiumsulfate, filtered and concentrated under reduced pressure. The obtainedresidue was azeotropically distilled 3 times with toluene (10 ml) toquantitatively give a triethylamine salt of the title compound (7.15 g)as a white solid.

(2) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-O-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinate[5′-O-DMTr-dT-suc-TPB]

The compound synthesized in Example 1-(1) (6.55 g, 8.78 mmol) and3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl alcohol (5.19g, 5.16 mmol) synthesized in Preparation Example 7 were dissolved inanhydrous dichloromethane (15 ml), 2-(1H-benzotriazol-1-yl)1,1,3,3-tetramethyluronium hexafluorophosphate [HBTU] (11.8 g, 30.8mmol) and N,N-diisopropylethylamine (5.53 ml, 31.2 mmol) were added, andthe mixture was stirred at room temperature for 1 hr. The mixture waswashed with saturated aqueous sodium hydrogen carbonate solution andsaturated brine, and the obtained organic layer was dried over sodiumsulfate and filtered. The filtrate was concentrated under reducedpressure and the obtained residue was purified by silica gel columnchromatography (hexane/ethyl acetate, 1% v/v triethylamine) to give thetitle compound (3.83 g, 45.3%) as a viscous solid.

Example 2 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinamate[5′-O-DMTr-dT-suc-NH-TPB]

Using a triethylamine salt (1.45 g, 1.94 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate, and3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl amine (1.02 g,1.10 mmol) synthesized in Preparation Example 8, and in the same manneras in Example 1-(2), the title compound (1.22 g, 72.4%) was obtained asa viscous solid.

Example 3 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(3,7,11,15-tetramethyl-1-hexadecanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^(Phy)-PA]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.46 g, 2.00 mmol) was dissolved in anhydrous tetrahydrofuran (10 ml),N,N-diisopropylethylamine (720 μl, 4.00 mmol) and the compoundsynthesized in Preparation Example 18 (990 mg, 3.00 mmol) were added,and the mixture was stirred at room temperature for 3 hr. Aftercompletion of the reaction, ethyl acetate (60 ml) and 5% aqueous sodiumhydrogen carbonate solution (15 ml) were added to the reaction mixtureto allow phase separation, and the aqueous phase was extracted withethyl acetate (30 ml). The combined organic phase was dried overanhydrous sodium sulfate, and the solvent was evaporated under reducedpressure. The concentrated oil (2.70 g) was purified by chromatography(silica gel; 50 g, eluate; 1% triethylamine-containing 10:1→1:1hexane-ethyl acetate) to give the title compound (1.08 g, 52.6%) as acolorless oil.

¹H-NMR (400 MHz, CDCl₈): δ0.84 (d, 6H, J=6.6 Hz), 0.86 (d, 6H, J=6.6Hz), 0.97 (d, 3H, J=6.6 Hz), 1.00-1.41 (m, 32H), 1.46-1.56 (m, 1H),1.94-2.18 (m, 2H), 2.21-2.33 (m, 1H), 2.34-2.46 (m, 2H), 2.62 (t, 1H,J=6.3 Hz), 2.67-2.83 (m, 1H), 3.34-3.67 (m, 5H), 3.69-3.86 (m, 1H), 3.80(s, 3H), 3.81 (s, 3H), 4.20-4.24 (m, 1H), 4.55-4.66 (m, 1H), 6.22-6.29(m, 1H), 6.82-6.87 (m, 4H), 7.10-7.14 (m, 1H), 7.21-7.33 (m, 7H),7.36-7.42 (m, 2H), 7.94 (brs, 1H), 8.17-8.29 (m, 1H)

³¹P-NMR (160 MHz, CDCl₃): δ 150.0, 150.6

m/z(ESI-MS): Anal. Calc. for C₅₉H₈₆N₅O₈P: 1023.6. Found 1022.3 (M−H)⁻

Example 4 Synthesis ofN³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine (1) Synthesisof 4-(3,7-dimethyl-1-octyloxy)benzoyl chloride

Under an argon atmosphere, 4-(3,7-dimethyl-1-octyloxy)benzoic acid wasdissolved in anhydrous chloroform (25 ml), and after ice-cooling,thionyl chloride (6.72 ml, 92.7 mmol) was added dropwise. The reactionmixture was stirred at room temperature overnight and concentrated underreduced pressure to give the title compound as an oil. The presentcompound was directly used for the next step.

(2) Synthesis of3′,5′-O-bis(trimethylsilyl)-N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]2′-deoxythymidine

2′-Deoxythymidine (5.00 g, 20.6 mmol) was azeotropically distilled 3times with anhydrous pyridine (10 ml), and under an argon atmosphere,the mixture was dissolved in anhydrous pyridine (60 ml),N,N-diisopropylethylamine (17.9 ml, 103 mmol) and trimethylsilylchloride (6.50 ml, 51.5 mmol) were added, and the mixture was stirred atroom temperature for 30 min. After stirring, an oil of theaforementioned 4-(3,7-dimethyl-1-octyloxy)benzoyl chloride was addeddropwise over 25 min, and thereafter the mixture was stirred at roomtemperature for 4 hr. The reaction mixture after completion of thereaction was ice-cooled, potassium dihydrogen phosphate (17 g) and water(80 ml) were added, and the mixture was stirred for 5 min. The mixturewas extracted with diethyl ether (100 ml), washed with saturated aqueouspotassium dihydrogen phosphate solution (50 ml) and saturated brine (50ml), the obtained organic layer was dried over sodium sulfate andfiltered, and the filtrate was concentrated under reduced pressure togive the title compound. The present compound was directly used for thenext step.

(3) Synthesis ofN³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine

3′,5′-O-bis-(trimethylsilyl)-N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidinewas dissolved in a mixed solvent of chloroform (70 ml) and methanol (70ml), trifluoroacetic acid (350 μl) was added, and the mixture wasstirred at room temperature for 30 min. After completion of thereaction, the solvent was evaporated and dissolved again in ethylacetate (150 ml). The mixture was washed with 5% aqueous sodium hydrogencarbonate solution (75 ml) and saturated brine (75 ml), and the organiclayer was dried over sodium sulfate and filtered. The solvent in thefiltrate was evaporated and the obtained residue was purified by silicagel column chromatography (dichloromethane/methanol) to give the titlecompound (7.10 g, 68.5%) as a white solid.

Example 5 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine

Under an argon atmosphere,N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine (7.10 g, 14.1mmol) was azeotropically distilled 3 times with anhydrous pyridine (10ml) and dissolved in anhydrous pyridine (130 ml). 4,4′-Dimethoxytritylchloride (4.83 g, 14.3 mmol) was added, and the mixture was stirredovernight. Water (130 ml) was added to the reaction mixture aftercompletion of the reaction, and the mixture was extracted with diethylether (260 ml) and washed with water (130 ml). The organic layer wasdried over sodium sulfate and filtered, the filtrate was concentrated,and the obtained residue was purified by silica gel columnchromatography (dichloromethane/methanol, 1% v/v triethylamine) to givethe title compound (10.2 g, 90.1%) as a white solid.

Example 6 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dT^((4-Cit-Bz))-PA]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-N³-[4-(3,7-dimethyl-1-octyloxy)benzoyl]-2′-deoxythymidine(6.47 g, 8.03 mmol) was azeotropically distilled 3 times with anhydrousacetonitrile (10 ml) and dissolved in anhydrous dichloromethane (40 ml).Under ice-cooling, N,N-diisopropylethylamine (5.60 ml, 32.0 mmol) wasadded, and a solution ofchloro-2-cyanoethyl-N,N-diisopropylphosphoramidite (2.36 ml, 10.0 mmol)in dichloromethane (40 ml) was added dropwise over 20 min. The reactionmixture was stirred at room temperature for 30 min and concentratedunder reduced pressure, and the obtained residue was purified by silicagel column chromatography (hexane/ethyl acetate, 3% v/v triethylamine)to give the title compound (7.32 g, 91.0%) as a white solid.

Example 7 Synthesis of5′-O-(4,4′-dimethoxytrityl)-3′-O-levulinoyl-N³-(3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl-2′-deoxythymidine

Under an argon atmosphere, potassium carbonate (2.10 g, 15.2 mmol) wassuspended in anhydrous N,N-dimethylformamide (50 ml), the compoundsynthesized in Preparation Example 19 (3.65 g, 10.1 mmol) and5′-O-(4,4′-dimethoxytrityl)-3′-O-levulinoyl-2′-deoxythymidine (3.25 g,5.06 mmol) were dissolved therein, and the mixture was stirred at 40° C.for 24 hr. The reaction mixture was filtered, water (50 ml) was added,and the mixture was extracted twice with diethyl ether (100 ml). Theorganic layer was washed with water (100 ml), dried over sodium sulfate,filtered and concentrated under reduced pressure. The obtained residuewas purified by silica gel column chromatography (dichloromethane, 1%v/v triethylamine) to give the title compound (4.80 g, 98%) as apale-yellow viscous solid.

Example 8 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-(3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl-2′-deoxythymidine

The compound synthesized in Example 7 (4.80 g, 4.96 mmol) was dissolvedin a mixed solvent of pyridine (40 ml) and acetic acid (10 ml),anhydrous hydrazine (244 μl, 7.71 mmol) was added, and the mixture wasstirred at room temperature for 30 min. Acetylacetone (1.07 ml, 10.3mmol) was added and the mixture was stirred at room temperature for 5min. Diethyl ether (100 ml) was added, and the mixture was washedsuccessively with 10% aqueous hydrogen sulfate potassium solution (50ml), 10% aqueous sodium hydrogen carbonate solution (50 ml) andsaturated brine (50 ml). The organic layer was concentrated to give thetitle compound (3.58 g, 83.0%) as a pale-yellow viscous solid.

Example 9 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-(3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl-2′-deoxythymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dT^(PhyOM)-PA]

Under an argon atmosphere, the compound synthesized in Example 8 (3.58g, 4.12 mmol) was azeotropically distilled 3 times with anhydrousacetonitrile (10 ml) and dissolved in anhydrous dichloromethane (22 ml).N,N-diisopropylethylamine (2.49 ml, 16.5 mmol) was added dropwise, andchloro-2-cyanoethyl-N,N-diisopropylphosphoramidite (1.15 ml, 5.15 mmol)was dissolved in anhydrous dichloromethane (22 ml) was added dropwiseover 20 min. After stirring at room temperature for 30 min, the mixturewas concentrated under reduced pressure and the obtained residue waspurified by silica gel column chromatography (hexane/ethyl acetate=8/2,3% v/v triethylamine) to give the title compound (3.90 g, 88.6%) as apale-yellow viscous solid.

Example 10 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-(3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl-2′-deoxythymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dr^(PhyOM)-PA]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.00 g, 1.34 mmol) was dissolved in anhydrous N,N-dimethylformamide (15ml), potassium carbonate (279 mg, 2.02 mmol) and(3,7,11,15-tetramethyl-1-hexadecanoyloxy)methyl chloride (970 mg, 2.69mmol) were added, and the mixture was stirred at room temperature for 16hr. The reaction mixture was filtered, water (20 ml) was added and themixture was extracted with diethyl ether (50 ml). The organic layer wasdried over sodium sulfate and filtered, the filtrate was concentratedunder reduced pressure, and the obtained residue was purified by silicagel column chromatography (ethyl acetate/hexane, 3% v/v triethylamine)to give the title compound (929 mg, 65.0%) as a pale-yellow viscoussolid.

Example 11 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N²-(3,7,11,15-tetramethyl-1-hexadecanoyl)-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dG^(Phy)-PA]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.52 g, 1.98 mmol) was dissolved in anhydrous tetrahydrofuran (20 ml),N,N-diisopropylethylamine (696 μl, 4.00 mmol) was added, and3,7,11,15-tetramethyl-1-hexadecanoyl chloride (993 mg, 3.00 mmol)synthesized in Preparation Example 18 was added dropwise. After stirringat room temperature for 1.5 hr, the reaction mixture was concentratedand the obtained residue was purified by silica gel columnchromatography (dichloromethane/methanol, 1% v/v triethylamine) to givethe title compound (1.73 g, 82.0%) as a pale-yellow viscous solid.

Example 12 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁶-(3,7,11,15-tetramethyl-1-hexadecanoyl)-2′-deoxyadenosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dA^(Phy)-PA]

Using5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.76 g, 2.33 mmol) and in the same manner as in Example 11, the titlecompound (1.12 g, 45.8%) was obtained as a pale-yellow viscous solid.

Example 13 Synthesis ofdeoxythymidinyl-[3→5′]-deoxythymidinyl-[3′→5′]-deoxythymidine(5′-d[TTT]-3′) (1) Synthesis of 5′-O-DMTr-dT^(PhyOM)-dT-suc-NH-TPB

Under an argon atmosphere, 5′-O-DMTr-dT-suc-NH-TPB (206 mg, 127 μmol)synthesized in Example 2 was dissolved in a mixed solvent of anhydrousheptane (650 μl) and anhydrous toluene (650 μl), trifluoroacetic acid(26.0 μl, 350 μmol) and 1H-pyrrole (17.5 μl, 254 μmol) were added, andthe mixture was stirred for 5 min. The completion of the deprotectionwas confirmed by thin layer chromatography, pyridine (28.3 μl, 350 μmol)and N-methylimidazole (13.9 μl, 175 μmol) were added, and the mixturewas stirred for 5 min. To the reaction mixture after neutralization wasadded the compound synthesized in Example 10 (271 mg, 254 μmol)dissolved in 0.25 mol/l 5-(benzylthio)-1H-tetrazole/acetonitrilesolution (1.0 ml), and the mixture was stirred for 10 min. 0.2 mol/lIodine pyridine/tetrahydrofuran/water=49/49/2 solution (1.27 ml) wasadded and the mixture was stirred for 5 min. To the reaction mixtureafter completion of the reaction was added heptane (5.0 ml) to allowphase separation. The lower layer was extracted, washed with a mixedsolution of acetonitrile (1.0 ml) and water (80 μl), and the obtainedorganic layer was concentrated under reduced pressure to give the titlecompound (291 mg, 99.5%) as a viscous solid.

(2) Synthesis of 5′-O-DMTr-dT^(PhyOM)-dT^(PhyOM)-dT-suc-NH-TPB

Using the compound synthesized in Example 13-(1) (291 mg, 126 μmol) andthe compound synthesized in Example 10 (271 mg, 254 μmol), and in thesame manner as in Example 13-(1), the title compound (369 mg, 98.0%) wasobtained as a viscous solid.

(3) Synthesis ofdeoxythymidinyl-[3′→5′]-deoxythymidinyl-[3′→5′]-deoxythymidine(5′-d[TTT]-3′)

The compound synthesized in Example 13-(2) and a solution (4.0 ml) of28% aqueous ammonia solution:40% aqueous methylamine solution=1:1 wereplaced in an autoclave, the mixture was heated at 65° C. for 16 hr, andconcentrated by a rotary evaporator under reduced pressure. The mixturewas adsorbed to C-18 reversed-phase cartridge column and washed with 0.1mol/L aqueous ammonium acetate solution. A dimethoxytrityl group bondedto the hydroxyl group at the 5′-terminal was deprotected with 2% aqueoustrifluoroacetic acid solution and eluted with 20% aqueous acetonitrilesolution to give the title compound.

m/z(ESI-MS): Anal. Calc. for C₃₀H₄₀N₆O₁₉P₂: 850.2. Found 849.1 (M−H)⁻

Example 14 Synthesis ofdeoxythymidinyl-[3′→5′]-deoxythymidinyl-[3′→5′]-deoxythymidine(5′-d[TTT]-3′) (1) Synthesis of 5′-O-DMTr-dT^((4-Cit-Bz))-dT-suc-NH-TPB

Using 5′-O-DMTr-dT-suc-NH-TPB synthesized in Example 2 (206 mg, 127μmol) and the compound synthesized in Example 6 (256 mg, 254 μmol), andin the same manner as in Example 13-(1), the title compound (273 mg,96.0%) was obtained as a viscous solid.

(2) Synthesis of 5′-O-DMTr-dT^((4-Cit-Bz))-dT^((4-Cit-Bz))-dT-suc-NH-TPB

Using the compound synthesized in Example 14-(1) (273 mg, 122 μmol) andthe compound synthesized in Example 6 (256 mg, 254 μmol), and in thesame manner as in Example 13-(1), the title compound (339 mg, 97.5%) wasobtained as a viscous solid.

(3) Synthesis ofdeoxythymidinyl-[3′→5′]-deoxythymidinyl-[3′→5′]-deoxythymidine(5′-d[TTT]-3′)

Using the compound synthesized in Example 14-(2) and in the same manneras in Example 13-(3), the title compound was obtained.

m/z(ESI-MS): Anal. Calc. for C₃₀H₄₀N₆O₁₉P₂: 850.18. Found 849.1 (M−H)⁻

Example 15 Synthesis ofdeoxyadenylyl-[3′→5′]-deoxyadenylyl-[3′→5′]-deoxythymidine(5′-d[AAT]-3′) (1) Synthesis of 5′-O-DMTr-dA^(Phy)-dT-suc-NH-TPB

Under an argon atmosphere, 5′-O-DMTr-dT-suc-NH-TPB (213 mg, 131 μmol)synthesized in Example 2 was dissolved in a mixed solvent of anhydrousheptane (700 μl) and anhydrous toluene (700 μl), trifluoroacetic acid(28.0 μl, 377 μmol) and 1H-pyrrole (18.1 μl, 262 μmol) were added, andthe mixture was stirred for 5 min. The completion of the deprotectionwas confirmed by thin layer chromatography, pyridine (30.5 μl, 377 μmol)and N-methylimidazole (15.0 μl, 189 μmol) were added, and the mixturewas stirred for 5 min. To the reaction mixture after neutralization wasadded the compound synthesized in Example 12 (275 mg, 262 μmol)dissolved in a mixed solvent of 0.25 mol/l5-(benzylthio)-1H-tetrazole/acetonitrile solution (1.0 ml) and anhydroustoluene (300 μl), and the mixture was stirred for 10 min. 5.78 mol/ltert-Butyl hydroperoxide/nonane solution (45.3 μl) was added and themixture was stirred for 5 min. To the reaction mixture after completionof the reaction was added heptane (4.0 ml) to allow layer separation.The lower layer was extracted and washed with a mixed solution ofacetonitrile (1.0 ml) and water (80 μl), and the obtained organic layerwas concentrated under reduced pressure to give the title compound (301mg) as a viscous solid quantitatively.

(2) Synthesis of 5′-O-DMTr-dA^(Phy)-dA^(Phy)-dT-suc-NH-TPB

Using the compound synthesized in Example 15-(1) (301 mg, 131 μmol) andthe compound synthesized in Example 12 (275 mg, 262 μmol), and in thesame manner as in Example 15-(1), the title compound (387 mg) wasobtained as a viscous solid quantitatively.

(3) Synthesis ofdeoxyadenylyl-[3′→5′]-deoxyadenylyl-[3′-35′]-deoxythymidine(5′-d[AAT]-3′)

The compound synthesized in Example 15-(2) and 28% aqueous ammoniasolution (4.0 ml) were placed in an autoclave, the mixture was heated at65° C. for 16 hr, and concentrated by a rotary evaporator under reducedpressure. The concentrated solution was adsorbed to C-18 reversed-phasecartridge column and washed with 0.1 mol/L aqueous ammonium acetatesolution. A dimethoxytrityl group bonded to the hydroxyl group at the5′-terminal was deprotected with 2% aqueous trifluoroacetic acidsolution and eluted with 20% aqueous acetonitrile solution to give thetitle compound.

m/z(ESI-MS): Anal. Calc. for C₃₀H₃₈N₁₂O₁₅P₂: 868.21. Found 867.1 (M−H)⁻

Example 16 Synthesis ofdeoxyguanosinyl-[3′→5′]-deoxyguanosinyl-[3′→5′]-deoxythymidine(5′-d[GGT]-3′) (1) Synthesis of 5′-O-DMTr-dG^(Phy)-dT-suc-NH-TPB

Using 5′-O-DMTr-dT-suc-NH-TPB (201 mg, 124 μmol) synthesized in Example2 and the compound (266 mg, 250 μmol) synthesized in Example 11, and inthe same manner as in Example 15-(1), the title compound (303 mg) wasobtained as a viscous solid quantitatively.

(2) Synthesis of 5′-O-DMTr-dG^(Phy)-dG^(Phy)-dT-suc-NH-TPB

Under an argon atmosphere, using the compound synthesized in Example16-(1) (303 mg, 124 μmol) and the compound synthesized in Example 11(265 mg, 249 μmol), and in the same manner as in Example 15-(1), thetitle compound (400.0 mg) was obtained as a viscous solidquantitatively.

(3) Synthesis ofdeoxyguanosinyl-[3′→5′]-deoxyguanosinyl-[3′→5′]-deoxythymidine(5′-d[GGT]-3′)

Using the compound synthesized in Example 16-(2) (23.8 mg, 7.37 mmol)and in the same manner as in Example 15-(3), the title compound wasobtained.

m/z(ESI-MS): Anal. Calc. for C₃₀H₃₈N₁₂O₁₇P₂: 900.20. Found 900.8 (M+H)⁺

Example 17 Synthesis ofdeoxycytidinyl-[3′-5′]-deoxycytidinyl-[3′→5′]-deoxythymidine(5′-d[CCT]-3′) (1) Synthesis of 5′-O-DMTr-dC^(Phy)-dT-suc-NH-TPB

Under an argon atmosphere, using 5′-O-DMTr-dT-suc-NH-TPB (203 mg, 125μmol) synthesized in Example 2 and the compound synthesized in Example 3(260 mg, 254 μmol), and in the same manner as in Example 15-(1), thetitle compound (366.9 mg) was obtained as a viscous solidquantitatively.

(2) Synthesis of 5′-O-DMTr-dC^(Phy)-dC^(Phy)-dT-suc-NH-TPB

Under an argon atmosphere, using the compound synthesized in Example17-(1) (367 mg, 125 μmol) and the compound synthesized in Example 3 (262mg, 256 μmol), and in the same manner as in Example 15-(1), the titlecompound (394 mg) was obtained as a viscous solid quantitatively.

(3) Synthesis ofdeoxycytidinyl-[3′→5′]-deoxycytidinyl-[3′→5′]-deoxythymidine(5′-d[CCT]-3′)

Using the compound synthesized in Example 17-(2) (20.8 mg, 6.59 mmol)and in the same manner as in Example 13-(3), the title compound wasobtained.

m/z(ESI-MS): Anal. Calc. for C₂₈H₃₈N₈O₁₇P₂: 820.2. Found 819.1 (M−H)⁻

Example 18 Synthesis of5′-O-DMTr-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhYOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT^(PhyOM)-dT-suc-NH-TPB

-   (1) Under an argon atmosphere, 5′-O-DMTr-dT-suc-NH-TPB (208 mg, 128    μmol) synthesized in Example 2 was dissolved in a mixed solvent of    anhydrous heptane (650 μl) and anhydrous toluene (650 μl),    trifluoroacetic acid (26.0 μl, 350 μmol) and 1H-pyrrole (17.7 μl,    256 μmol) were added, and the mixture was stirred for 5 min. The    completion of the deprotection was confirmed by thin layer    chromatography, pyridine (28.3 μl, 350 μmol) and N-methylimidazole    (13.9 μl, 175 μmol) were added, and the mixture was stirred for 5    min. To the reaction mixture after neutralization was added the    compound synthesized in Example 9 (273 mg, 256 μmol) dissolved in    0.25 mol/l 5-(benzylthio)-1H-tetrazole/acetonitrile solution (1.0    ml), and the mixture was stirred for 10 min. 5.78 mol/l tert-Butyl    hydroperoxide/nonane solution (383 μmol, 66.3 μl) was added and the    mixture was stirred for 5 min. The reaction mixture after completion    of the reaction was partitioned by adding heptane (3.0 ml), the    lower layer was extracted, washed with a mixed solution of    acetonitrile (1.0 ml) and water (80 μl), and the obtained organic    layer was concentrated under reduced pressure to give a compound    same as that synthesized in Example 14-(1) (291 mg, 98.8%) as a    viscous solid.-   (2) By repeating a similar operation 18 times, the title compound    (1.67 g, 89.6%) was obtained as an orange solid.

Example 19 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(2-ethyl-1-hexanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((2Et-Hex))-PA]

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](20.4 g, 28.0 mmol) was dissolved in tetrahydrofuran (200 ml),N,N-diisopropylethylamine (7.4 g, 57.0 mmol) and 2-ethyl-1-hexanoicanhydride (11.4 g, 42.0 mmol) were added, and the mixture was stirred atroom temperature for 3 days. To the reaction mixture after completion ofthe reaction were added ethyl acetate and water, and the mixture wasextracted. The ethyl acetate layer was washed with 5% aqueous sodiumhydrogen carbonate solution and saturated brine. The organic layer wasdried over anhydrous sodium sulfate, the solvent was evaporated underreduced pressure, and the obtained crude product was purified by silicagel column chromatography (eluate; 1% triethylamine-containing 10:1→1:1heptane-ethyl acetate) to give the title compound (15.9 g, 66.5%).

Example 20 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(3,5,5-trimethyl-1-hexanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((3,5,5-Me3Hex))-PA] (1) Synthesis ofN⁴-(3,5,5-trimethyl-1-hexanoyl)-2′-deoxycytidine

Suspending 2′-Deoxycytidine (2.3 g, 10.0 mmol) in dry pyridine, followedby concentration under reduced pressure was repeated 3 times to performdehydrative azeotropic distillation, the concentrate was suspended againin dry pyridine (60 ml), and trimethylsilyl chloride (6.4 ml, 50 mmol)was added dropwise over 5 min. To the reaction mixture was added3,5,5-trimethyl-1-hexanoyl chloride (9.5 ml, 50.0 mmol) over 5 min.After completion of the reaction, under ice-cooling, aqueous ammonia (25ml) was added and the mixture was reacted for 20 min. The reactionmixture was concentrated under reduced pressure. Water (150 ml) wasadded to the concentrate, and the mixture was extracted 3 times withethyl acetate (100 ml). The organic layer was concentrated and theobtained oil was purified by silica gel column chromatography to givethe title compound (4.7 g).

(2) Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(3,5,5-trimethyl-1-hexanoyl)-2′-deoxycytidine

Dissolving the compound obtained in Example 20-(1) (4.7 g) in drypyridine, followed by concentration under reduced pressure was repeated3 times to perform dehydrative azeotropic distillation, and theconcentrate was dissolved in dry pyridine (40 ml). 4,4′-Dimethoxytritylchloride (3.8 g, 11.0 mmol) was added and the mixture was stirred for 30min. Water (100 ml) was added to the reaction mixture and the mixturewas extracted 3 times with ethyl acetate (100 ml). The organic layer wasfurther washed with water and concentrated under reduced pressure, andthe obtained oil was purified by silica gel column chromatography togive the title compound (5.2 g, 77.3%).

(3) Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(3,5,5-trimethyl-1-hexanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((3,5,5-Me3Hex))-PA]

Dissolving the compound obtained in Example 20-(2) in dry acetonitrile(5 ml), followed by concentration under reduced pressure was repeated 3times to perform dehydrative azeotropic distillation and the concentratewas dissolved in dry dichloromethane (30 ml). N,N-diisopropylethylamine(5.4 ml, 30.9 mmol) was added dropwise over 5 min, and a solution ofchloro-2-cyanoethyl-N,N-diisopropylphosphoramidite (2.2 ml, 9.7 mmol) indichloromethane (30 ml) was added dropwise over 15 min. After reactionat room temperature for 30 min, the reaction mixture was concentratedunder reduced pressure, and the obtained oil was purified by silica gelcolumn chromatography (dichloromethane/methanol/triethylamine=94/3/3).The fractions containing the object product were concentrated to drynessto give the title compound (3.7 g, 55.3%) as a white solid.

Example 21 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(2,2,4,8,10,10-hexamethyl-5-dodecanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((Me6Dodecanoyl))-PA]

According to the method described in Example 19, the title compound(16.5 g, 47.3%) was prepared from5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](25.6 g, 35.1 mmol) using 2,2,4,8,10,10-hexamethyl-5-dodecanoyl chloride(16.5 g, 54.5 mmol) as an acylating agent.

Example 22 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(2-heptyl-1-undecanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((2-HepUndecanoyl))-PA]

According to the method described in Example 19,5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.8 g, 2.5 mmol) was reacted with 2-heptyl-1-undecanoic anhydride (2.1g, 3.8 mmol) as an acylating agent. Since the reaction proceeds slowly,2-heptyl-1-undecanoyl chloride (1.2 g, 3.8 mmol) was added again as anacylating agent, and the mixture was reacted for 30 min. Aftercompletion of the reaction, the mixture was extracted with chloroform,and the organic layer was washed with 5% aqueous sodium hydrogencarbonate solution, concentrated and purified by silica gel columnchromatography to prepare the title compound (1.7 g, 67.5%).

Example 23 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-(2-hexyl-1-decanoyl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^((2-HexDecanoyl))-PA]

According to the method described in Example 19, the title compound(12.6 g, 47.0%) was prepared from5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](20.2 g, 28.0 mmol), using 2-hexyl-1-decanoylchloride (11.4 g, 41.0mmol) as an acylating agent.

Example 24 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N⁴-n-tetradecanoyl-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dC^(Myr)-PA]

According to the method described in Example 19, the title compound (1.3g, 49.2%) was prepared from5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](2.0 g, 2.7 mmol), using the corresponding tetradecanoyl chloride (751mg, 3.0 mmol) as an acylating agent.

Example 25 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N²-[bis(3,7-dimethyl-octyl)amino-methylene]-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite][5′-O-DMTr-dG^((N,N-Cit2-methylene))-PA]

Suspending5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1.0 g, 1.3 mmol) obtained in Preparation Example 22 in dry pyridine,followed by concentration under reduced pressure was repeated twice, theconcentrate was further dissolved in toluene, concentrated under reducedpressure, and subjected to dehydrative azeotropic distillation.Thereafter, in dry methanol (2.6 ml), the concentrate was reacted withthe compound synthesized in Preparation Example 23 overnight andconcentrated under reduced pressure, and the obtained oil was purifiedby silica gel column chromatography to give the title compound (184 mg,13.1%).

Example 26 Synthesis ofdA^(Bz)-dC^(Bz)-dA^(Bz)-dT-dG^(ibu)-dC^(Bz)-dA^(Bz)-dT-dT-suc-NH-TPB (1)Synthesis of dT-dT-suc-NH-TPB

Under an argon atmosphere, 5′-O-DMTr-dT-suc-NH-TPB (225 mg, 139 μmol)was dissolved in anhydrous cyclopentyl methyl ether (1.0 ml), and1H-pyrrole (9.6 μl, 139 μmol) was added. Furthermore, 20 μl of asolution (2.0 ml) of trifluoromethanesulfonic acid (24.4 μl, 2.78 μmol)in cyclopentyl methyl ether, prepared separately, was added, and themixture was stirred at room temperature for 10 min. The completion ofthe deprotection was confirmed by thin layer chromatography, and themixture was neutralized with a solution (14.0 μl, 176 μmol) of 0.2 mol/lN-methylimidazole in cyclopentyl methyl ether. dT-CE Phosphoramidite(5′-O-(4,4′-dimethoxytrityl)deoxythymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(207 mg, 278 μmol) dissolved in 0.3 mol/l4,5-dicyanoimidazole/acetonitrile solution was added and the mixture wasstirred for 10 min. Thereafter, pH 6.8 phosphoric acid buffer (1.0 ml),aqueous hydrogen peroxide (47 μl, 417 μmol) and potassium iodide (23.1mg, 139 μmol) were added to the reaction mixture, and the mixture wasstirred for 15 min. The reaction mixture was washed with 10% aqueoussodium thiosulfate solution. To the obtained organic layer were added1H-pyrrole (9.6 μl, 139 μmol) and trifluoroacetic acid (206 μl, 2.8mmol) and the mixture was stirred for 10 min. The organic layer waswashed with 10% aqueous potassium hydrogen sulfate solution, 10% aqueoussodium hydrogen carbonate solution and brine. The obtained organic layerwas concentrated under reduced pressure to give the title compound (316mg) as a viscous solid quantitatively.

(2) Synthesis of dA^(Bz)-dT-dT-suc-NH-TPB

The compound obtained in Example 26-(1) (316 mg) was dissolved indichloromethane (2.0 ml), dA-CE phosphoramidite(5′-O-(4,4′-dimethoxytrityl)-N⁶-benzoyl-2′-deoxyadenosine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(239 mg, 278 μmol) dissolve in 0.3 mol/l4,5-dicyanoimidazole/acetonitrile solution was added, and the mixturewas stirred for 10 min. Thereafter, pH 6.8 phosphoric acid buffer (1.0ml), potassium iodide (20.0 mg, 97.4 μmol) and aqueous hydrogen peroxide(16.0 μl, 139 μmol) were added to the reaction mixture, and the mixturewas stirred for 15 min. The reaction mixture was washed with 10% aqueoussodium thiosulfate solution. To the obtained organic layer were added1H-pyrrole (9.6 μl, 139 μmol) and trifluoroacetic acid (206 μl, 2.8mmol) and the mixture was stirred for 10 min. The organic layer waswashed with 10% aqueous potassium hydrogen sulfate solution, 10% aqueoussodium hydrogen carbonate solution and brine. The obtained organic layerwas concentrated under reduced pressure to give the title compound (442mg) as a viscous solid quantitatively.

(3) Synthesis ofdA^(Bz)-dC^(Bz)-dA^(Bz)-dT-dG^(ibu)-dC^(Bz)-dA^(Bz)-dT-dT-suc-NH-TPB

The title compound was synthesized by repeating the method described inExample 26-(2).

Example 27 Synthesis of5′-O-DMTr-dC^((2Et-Hex))-dC^((2Et-Hex))-dT-suc-NH-TPB (1) Synthesis of5′-O-DMTr-dC^((2Et-Hex))-dT-suc-NH-TPB

Using 5′-O-DMTr-dT-suc-NH-TPB (207 mg, 127 μmol) and the phosphoramiditemonomer synthesized in Example 19 (336 mg, 393 μmol), and in the samemanner as in Example 15-(1), the title compound (226 mg, 84.9%) wasobtained as a viscous solid.

(2) Synthesis of 5′-O-DMTr-dC^((2Et-Hex))-dC^((2Et-Hex))-dT-suc-NH-TPB

Under conditions similar to those of Example 27-(1), cytidine derivativewas elongated by further using the phosphoramidite monomer synthesizedin Example 19 to give the title compound (249 mg, 90.0%).

thin layer chromatography: Rf 0.18 (eluent=ethyl acetate)

Example 28 Synthesis of5′-O-DMTr-dC^((3,5,5-Me3Hex))-dC^((3,5,5-Me3Hex))-dC^((3,5,5-Me3Hex))-dC^((3,5,5-Me3Hex))-dT-suc-NH-TPB

Based on the method described in Example 27, the title compound (314 mg,72.2%) was obtained from 5′-O-DMTr-dT-suc-NH-TPB (198.6 mg, 122 μmol) byrepeating elongation of cytidine derivative 4 times using thephosphoramidite monomer described in Example 20.

thin layer chromatography: Rf 0.21 (eluent=toluene/acetonitrile=9/1(v/v))

Example 29 Synthesis of5′-O-DMTr-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dC^((2-HepUndecanoyl))-dT-suc-NH-TPB

Based on the method described in Example 27, the title compound (210 mg,49.1%) was obtained from 5′-O-DMTr-dT-suc-NH-TPB (203 mg, 125 μmol) byrepeating elongation of cytidine derivative 9 times using thephosphoramidite monomer described in Example 22.

thin layer chromatography: Rf 0.13 (eluent=ethyl acetate)

Example 30 Synthesis of5′-O-DMTr-dC^(Myr)-dC^(Myr)-dC^(Myr)-dC^(Myr)-dT-suc-NH-TPB

Based on the method described in Example 27, the title compound (199 mg,82.2%) was obtained from 5′-O-DMTr-dT-suc-NH-TPB (102 mg, 63.1 μmol) byrepeating elongation of cytidine derivative 4 times using thephosphoramidite monomer described in Example 24.

thin layer chromatography: Rf 0.14 (eluent=ethyl acetate)

Example 31 Synthesis of5′-O-DMTr-dC^((Me6Dodecanoyl))-dC^((Me6Dodecanoyl))-dC^((Me6Dodecanoyl))-dC^((Me6Dodecanoyl))-dC^((Me6Dodecanoyl))-dC^(Phy)-dC^(Phy)-dC^(Phy)-dC^(Phy)-dT-suc-NH-TPB

Based on the method described in Example 27,5′-O-DMTr-dC^(Phy)-dC^(Phy)-dC^(Phy)-dC^(Phy)-dT-suc-NH-TPB (534 mg,97.9%) was obtained from 5′-O-DMTr-dT-suc-NH-TPB (210 mg, 129 μmol) byrepeating elongation of cytidine derivative 4 times using5′-O-DMTr-dC^(Phy)-PA. Furthermore, based on the method described inExample 27, the title compound (598 mg, 64.6%) was obtained by repeatingelongation of cytidine derivative 5 times using5′-O-DMTr-dC^((Me6Dodecanoyl))-PA described in Example 21.

Example 32 Synthesis of2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxycytidine-[3′→5′]-2′-deoxythymidine[5′-d(CCCCCCCCCT)-3′]

To the compound synthesized in Example 31 (20.0 mg, 2.8 μmol) were added40% aqueous methylamine solution (2.0 ml) and 28% aqueous ammonia (2.0ml), and the mixture was reacted in an autoclave at 65° C. for 1 hr. Thereaction mixture was concentrated with a rotary evaporator under reducedpressure, adsorbed on C-18 reversed-phase cartridge column, and washedwith 0.1 mol/l aqueous ammonium acetate solution. The dimethoxytritylgroup bonded to the 5′-terminal hydroxyl group was removed with aqueous2% trifluoroacetic acid solution, and the mixture was eluted with 20%aqueous acetonitrile solution to give the title compound.

IEX-HPLC (DNA Pac PA200 (4×250 mm)), flow rate 1 ml/min, eluent A 20 mMTris-HCl (pH 7.5), eluent B 400 mM NaClO₄/20 mM Tris-HCl (pH 7.5),gradient 20% to 70% for 30 min, λ=260 nm: RT=6.68 min (94.0 area %)

MALDI-TOF/MS: 2843.87 [M−H]⁻

Example 33 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using a triethylamine salt (3.52 g, 4.63 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate and2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl amine (2.92 g, 2.73 mmol)obtained in Preparation Example 24, and in the same manner as in Example1-(2), a filtrate of the reaction solution was obtained. Said filtratewas concentrated under reduced pressure and the obtained oil waspurified by silica gel column chromatography (hexane:ethylacetate=75:25-50/50 (v/v), containing 3% triethylamine) to give thetitle compound (3.60 g, 77.6%) as an oil.

Example 34 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[4,4′-bis(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using a triethylamine salt (2.88 g, 3.79 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate and4,4′-bis(2,3-dihydrophytyloxy)benzhydryl amine (1.73 g, 2.23 mmol)obtained in Preparation Example 25, and in the same manner as in Example1-(2), a filtrate of the reaction solution was obtained. Said filtratewas concentrated under reduced pressure and the obtained oil waspurified by silica gel column chromatography (hexane:ethylacetate=100:0-30/70 (v/v), containing 3% triethylamine) to give thetitle compound (1.87 g, 59.8%) as an oil.

Example 35 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[3,5-bis(2,3-dihydrophytyloxy)benzyl]succinamate

Using a triethylamine salt (2.84 g, 3.74 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate and3,5-bis(2,3-dihydrophytyloxy)benzyl amine (1.54 g, 2.20 mol) obtained inPreparation Example 26, and in the same manner as in Example 1-(2), afiltrate of the reaction solution was obtained. Said filtrate wasconcentrated under reduced pressure and the obtained oil was purified bysilica gel column chromatography (hexane:ethyl acetate=90:10-20/80(v/v), containing 1% triethylamine) to give the title compound (0.97 g,33%) as an oil.

Example 36 Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using the compound synthesized in Example 33 (199.0 mg, 117.1 μmol) anda dT-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)deoxythymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(223.8 mg, 0.30 mmol), and in the same manner as in Example 18-(1), thetitle compound (213.8 mg, 88.1%) was obtained as a viscous oil.

(2) Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′)

To the compound synthesized in Example 36-(1) (about 50 mg) was added amixture (5.0 ml) of 28% aqueous ammonia solution:40% aqueous methylaminesolution=1:1 (v/v), and the mixture was incubated at room temperaturefor 1 hr. The reaction mixture was concentrated in a rotary evaporator,the obtained crude product was adsorbed to C-18 reversed-phase cartridgecolumn and washed with 0.1 mol/l aqueous ammonium acetate solution. Thedimethoxytrityl group bonded to the 5′-terminal hydroxyl group wasremoved with aqueous 2% trifluoroacetic acid solution, and the mixturewas eluted with 20% aqueous acetonitrile solution to give the titlecompound.

m/z(ESI-MS): Anal. Calc. for C₂₀H₂₇N₄O₁₂P: 546.12. Found 545.1 (M−H)⁻

Example 37 Synthesis of 2′-methoxyuridinyl-[3′→5′]-2′-deoxythymidine(5′-U(M)dT-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-methoxyuridine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[2,3,4-tris(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using the compound synthesized in Example 33 (200.4 mg, 117.9 μmol) anda 2′-OMe-U-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-methoxyuridine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(222.3 mg, 0.30 mmol), and in the same manner as in Example 18-(1), thetitle compound (219.1 mg, 89.2%) was obtained as a viscous oil.

(2) Synthesis of 2′-methoxyuridinyl-[3′→5′]-2′-deoxythymidine(5′-U(M)dT-3′)

Using the compound synthesized in Example 37-(1) (about 50 mg) and inthe same manner as in Example 36-(2), the title compound was obtained.

m/z(ESI-MS): Anal. Calc. for C₂₀H₂₇N₄O₁₃P: 562.13. Found 561.1 (M−H)⁻

Example 38 Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[4,4′-bis(2,3-dihydrophytyloxy)benzhydryl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[4,4′-bis(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using the compound synthesized in Example 34 (200.1 mg, 0.14 mmol) and a2′-dT-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(267.3 mg, 0.36 mmol), and in the same manner as in Example 18-(1), thetitle compound (154.3 mg, 61.0%) was obtained as a viscous oil.

(2) Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′)

Using the compound synthesized in Example 38-(1) (about 50 mg) and inthe same manner as in Example 36-(2), the title compound was obtained.

m/z(ESI-MS): Anal. Calc. for C₂₀H₂₇N₄O₁₂P: 546.12. Found 545.1 (M−H)⁻

Example 39 Synthesis of 2′-methoxyuridinyl-[3′→5′]-2′-deoxythymidine(5′-U(M)dT-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[4,4′-bis(2,3-dihydrophytyloxy)benzhydryl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-methoxyuridine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[4,4′-bis(2,3-dihydrophytyloxy)benzhydryl]succinamate

Using the compound synthesized in Example 34 (201.7 mg, 0.14 mmol) and a2′-OMe-U-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-methoxyuridine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(278.9 mg, 0.37 mmol), and in the same manner as in Example 18-(1), thetitle compound (137.3 mg, 53.3%) was obtained as a viscous oil.

(2) Synthesis of 2′-methoxyuridinyl-[3′→5′]-2′-deoxythymidine(5′-U(M)dT-3′)

Using the compound synthesized in Example 39-(1) (about 50 mg) and inthe same manner as in Example 36-(2), the title compound was obtained.

m/z(ESI-MS): Anal. Calc. for C₂₀H₂₇N₄O₁₃P: 562.13. Found 561.1 (M−H)⁻

Example 40 Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[3,5-bis(2,3-dihydrophytyloxy)benzyl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[3,5-bis(2,3-dihydrophytyloxy)benzyl]succinamate

Using the compound synthesized in Example 35 (196.7 mg, 0.15 mmol) and a2′-dT-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(288.2 mg, 0.39 mmol), and in the same manner as in Example 18-(1), thetitle compound (76.7 mg, 30.5%) was obtained as a viscous oil.

(2) Synthesis of 2′-deoxythymidinyl-[3′→5′]-2′-deoxythymidine(5′-d[TT]-3′)

Using the compound synthesized in Example 40-(1) (about 50 mg) and inthe same manner as in Example 36-(2), the title compound was obtained.

m/z(ESI-MS): Anal. Calc. for C₂₀H₂₇N₄O₁₂P: 546.12. Found 545.1 (M−H)⁻

Example 41 Synthesis of2′-deoxy-2′-fluorouridinyl[3′→5′]-2′-deoxythymidine (5′-U(F)dT-3′) using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(2,3-dihydrophytyloxy)benzyl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(2,3-dihydrophytyloxy)benzyl]succinamate

Using the compound synthesized in Example 2 (202.4 mg, 0.13 mmol) andthe 2′-F-U-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(291.5 mg, 0.39 mmol) obtained in Preparation Example 27, and in thesame manner as in Example 18-(1), the title compound (198.9 mg, 78.8%)was obtained as a viscous oil.

(2) Synthesis of 2′-deoxy-2′-fluorouridinyl[3′→5′]-2′-deoxythymidine(5′-U(F)dT-3′)

Using the compound synthesized in Example 41-(1) (about 50 mg) and inthe same manner as in Example 36-(2), the title compound was obtained.

m/z (ESI-MS): Anal. Calc. for C₁₉H₂₄FN₄O₁₂P: 550.11. Found 549.1 (M−H)⁻

Example 42 Synthesis of2′-O,4′-C-methylenethymidinyl[3′->5′]-2′-deoxythymidine (5′-(LNA)TdT-3′)using5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(2,3-dihydrophytyloxy)benzyl]succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylenethymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(2,3-dihydrophytyloxy)benzyl]succinamate

Using the compound synthesized in Example 2 (197.8 mg, 0.13 mmol) and anLNA-T-CE phosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylenethymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(250.7 mg, 0.32 mmol), and in the same manner as in Example 18-(1), thetitle compound (220.3 mg, 88.3%) was obtained as a viscous oil.

(2) Synthesis of 2′-O,4′-C-methylenethymidinyl[3′→5′]-2′-deoxythymidine(5′-(LNA)TdT-3′)

To the compound synthesized in Example 42-(1) (about 50 mg) was added asolution (5.0 ml) of 10% (v/v) tert-butylamine dissolved in 28% aqueousammonia solution, and the mixture was heated to 65° C. in an autoclaveand reacted overnight. The reaction mixture was concentrated in a rotaryevaporator, the obtained crude product was adsorbed to C-18reversed-phase cartridge column, and washed with 0.1 mol/l aqueousammonium acetate solution. The dimethoxytrityl group bonded to the5′-terminal hydroxyl group was removed with aqueous 2% trifluoroaceticacid solution, and the mixture was eluted with 20% aqueous acetonitrilesolution to give the title compound.

m/z(ESI-MS): Anal. Calc. for C₂₁H₂₇N₄O₁₃P: 574.13. Found 573.1 (M−H)⁻

Example 43 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-[4-(2,3-dihydrophytyloxy)benzoyl]-2′-O,4′-C-methylenethymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite](1) Synthesis of 4-(2,3-dihydrophytyloxy)benzoic acid

In the same manner as in Preparation Example 20, the title compound wasprepared using 2,3-dihydrophytyl bromide and methyl (4-hydroxy)benzoatedescribed in Preparation Example 2.

¹H-NMR (400 MHz, CDCl₃): δ 0.83-0.98 (m, 15H), 1.01-1.92 (m, 24H),4.01-4.12 (m, 2H), 6.91-6.96 (m, 2H), 8.03-8.08 (m, 2H)

(2) Synthesis of 4-(2,3-dihydrophytyloxy)benzoyl chloride

The benzoic acid compound prepared in the above-mentioned Example 43-(1)was converted to the corresponding acid chloride in the same manner asin Example 4-(1) to give an oily compound. The present compound wasdirectly used for the next step.

(3) Synthesis of5′-O-(4,4′-dimethoxytrityl)-N³-[4-(2,3-dihydrophytyloxy)benzoyl]-2′-O,4′-C-methylenethymidine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

An operation of dissolving commercially available LNA-T-CEphosphoramidite reagent(5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylenethymidine-3′-[O-(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)(701.7 mg, 0.91 mmol) in anhydrous pyridine (5.0 ml), followed byconcentration under reduced pressure was repeated 3 times to performdehydrative azeotropic distillation of adhesive water. Thereafter, underan argon atmosphere, the reaction mixture was dissolved in dry pyridine(8.0 ml), N,N-diisopropylethylamine (0.17 ml, 1.12 mmol) and acidchloride (0.6 g, 1.36 mmol) prepared in the above-mentioned Example42-(2) were added, and the mixture was stirred at room temperature for 9hr. Ethyl acetate (50 ml) and water (10 ml) were added to the reactionmixture to allow layer separation. The organic layer was washed 3 timeswith 10% aqueous sodium hydrogen carbonate solution and once with brine(10 ml), and the obtained organic layer was dried over sodium sulfate.The filtered filtrate was concentrated and the obtained crude productwas purified by silica gel column chromatography (hexane:ethylacetate=100:0-30/70 (v/v), containing 1% triethylamine). The objectfractions were collected and concentrated, and the concentrated residuewas azeotropically distilled with toluene and dried to solidness to givethe title compound (0.77 g, 65.5%).

Example 44 Synthesis of2′-O,4′-C-methylenethymidinyl[3′→5′]-2′-deoxythymidine (5′-(LNA)TdT-3′)(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylenethymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-N-[3,4,5-tris(2,3-dihydrophytyloxy)benzyl]succinamate

Using the compound synthesized in Example 2 (200 mg, 0.12 mmol) and thephosphoramidite monomer synthesized in Example 43 (362 mg, 0.31 mmol),and in the same manner as in Example 18-(1), the title compound (295 mg,99.4%) was synthesized.

(2) Synthesis of 2′-O,4′-C-methylenethymidinyl[3′→5′]-2′-deoxythymidine(5′-(LNA)TdT-3′)

The compound (about 20 mg) synthesized in Example 44-(1) was treatedaccording to a method similar to that in Example 32, the title compoundwas obtained by deprotection and cutout from the anchor.

m/z(ESI-MS): Anal. Calc. for C₂₁H₂₇N₄O₁₃P: 574.13. Found 573.1 (M−H)⁻

Comparative Example 1 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-N²-isobutyryl-2′-deoxyguanosine-3′-[O-(2-cyanoethyl)]phosphoryl-N⁴-benzoyl-2′-deoxycytidine-3′-[O-(2-cyanoethyl)]phosphoryl-N⁶-benzoyl-2′-deoxyadenosine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-2′-deoxythymidin-3′-yl-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinate

Under an argon atmosphere,5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinate(509 mg, 313 μmol) was dissolved in a mixed solvent of anhydrous heptane(3.6 ml) and anhydrous toluene (3.6 ml), trifluoroacetic acid (279 μl,3.76 mmol) and 1H-pyrrole (217 μl, 3.13 mmol) were added, and themixture was stirred for 15 min. The completion of the deprotection wasconfirmed by thin layer chromatography, acetonitrile (1.2 ml), pyridine(304 μl, 3.76 mmol) and N-methylimidazole (149 μl, 1.88 mmol) wereadded, and the mixture was stirred for 5 min. To the reaction mixtureafter neutralization was added a solution (1.2 ml) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)(466 mg, 626 μmol) in acetonitrile, and the mixture was stirred for 1hr. 2,6-Lutidine (350 μl), N-methylimidazole (350 μl) and aceticanhydride (350 μl) were added and the mixture was stirred for 5 min.1.0M iodine pyridine/THF/H₂O solution (1.25 ml) was added and themixture was stirred at room temperature for 10 min. The reaction mixtureafter completion of the reaction was partitioned by adding heptane (7.2ml) and water (480 μl), the lower layer was extracted, and the heptanelayer was washed with acetonitrile containing water, and the obtainedorganic layer was concentrated under reduced pressure to give5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-[O-(2-cyanoethyl)]phosphoryl-deoxythymidin-3′-yl-[3,4,5-tris(3,7,11,15-tetramethyl-1-hexadecanyloxy)benzyl]succinate(620 mg, 99.8%).

According to the nucleic acid base sequence of the object product, asimilar operation was repeated using the corresponding commerciallyavailable phosphoramidite monomer to synthesize the title compound. Whendeoxyguanosine was elongated, the resultant product showed insufficientliposolubility, and the yield after extraction operation decreased to90%. When deoxythymidine was further elongated, it could not bedissolved in a heptane/toluene mixed solvent, and the yield afterreaction and extraction decreased to 85%. The following Tables showphosphoramidite monomers used in each stage and the yield thereof.

TABLE 1 Monomer product Yield 5′-O-(4,4′-dimethoxytrityl)-DMTr-dTT-suc-TPB 99.8% deoxythymidine-3′-O-(2- cyanoethyl-N,N-diisopropylphosphoramidite) N⁶-benzoyl-5′-O-(4,4′- DMTr-d(A^(Bz)TT)-suc-99.2% dimethoxytrityl)-deoxyadenosine- TPB 3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) N⁴-benzoyl-5′-O-(4,4′-DMTr-d(C^(Bz)A^(Bz)TT)- 98.3% dimethoxytrityl)-deoxycytidine- suc-TPB3′-O-(2-cyanoethyl-N,N- diisopropylphosphoramidite)N²-isobutyryl-5′-O-(4,4′- DMTr-d(G^(ibu)C^(Bz)A^(Bz)TT)- 90.3%dimethoxytrityl)-deoxyguanosine- suc-TPB 3′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) 5′-O-(4,4′-dimethoxytrityl)- DMTr- 85.2%deoxythymidine-3′-O-(2- d(TG^(ibu)C^(Bz)A^(Bz)TT)-suc- cyanoethyl-N,N-TPB diisopropylphosphoramidite)

Comparative Example 2 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-(3,4,5-tris(octadecyloxy)benzyl)succinamate(1) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate

Under an argon atmosphere, 5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine(5.00 g, 9.18 mmol), succinic anhydride (1.38 g, 13.8 mmol) andtriethylamine (3.85 mL, 27.5 mmol) were dissolved in dichloromethane (95mL), and the mixture was stirred at room temperature for 8 hr. Thecompletion of the reaction was confirmed by thin layer chromatography,and the reaction mixture was partitioned and washed three times with2.0M phosphoric acid-triethylamine buffer (pH 7.50). The organic layerwas evaporated under reduced pressure to give a triethylamine salt (7.02g, 98%) of 5′-O-(4,4′-dimethoxytrityl)deoxythymidine-3′-O-succinate as acolorless frothy solid.

(2) Synthesis of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidin-3′-yl-N-(3,4,5-tris(octadecyloxy)benzyl)succinamate

A triethylamine salt (1.45 g, 1.94 mmol) of5′-O-(4,4′-dimethoxytrityl)-2′-deoxythymidine-3′-O-succinate and3,4,5-tris(octadecyloxy)benzyl amine (1.02 g, 1.10 mmol) were dissolvedin anhydrous dichloromethane (15 mL),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate[HBTU] (2.53 g, 6.60 mmol) and N,N-diisopropylethylamine (1.17 mL, 6.60mmol) were added, and the mixture was stirred at room temperature for 1hr. The disappearance of the starting material was confirmed by thinlayer chromatography, and the mixture was washed with saturated aqueoussodium hydrogen carbonate solution and saturated brine. The organiclayer was dried over sodium sulfate and filtered, and the filtrate wasconcentrated under reduced pressure. Methanol was added to theconcentrated solution, the mixture was filtered, and the obtained solidwas purified by silica gel column chromatography(dichloromethane/methanol, 1% v/v triethylamine) to give the titlecompound (1.22 g, 72.4%) as a white solid.

In the following, Table 2 to Table 13 show the structural formulas andcompound data of the compounds produced in Preparation Examples 1 to 27,Examples 1, 2, 4 to 12, 19 to 25, 33 to 35, 43, and Comparative Example2.

TABLE 2 Prep. Ex. 1

¹H-NMR(300 MHz): δ 0.80-0.93(15H, m, Me), 0.98-1.70(24H, br, m,Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—OH), 3.62-3.75(2H, —CH₂ —OH) Prep. Ex. 2

¹H-NMR(300 MHz): δ 0.79-0.92(15H, m, Me), 0.95-1.95(24H, br, m,Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—Br), 3.35-3.52(2H, —CH₂ —Br) Prep. Ex. 3

¹H-NMR(300 MHz): δ 1.09-1.43(m, 24H), 1.48-1.66(m, 5H), 3.63-3.70(m, 2H)Prep. Ex. 4

¹H-NMR(300 MHz): δ 1.12-1.43(m, 24H), 1.48-1.70(m, 4H), 1.84-1.90(m,1H), 3.36-3.49(m, 2H) Prep. Ex. 5

2-chloro-5-(2,3-dihydrophytyloxy)benzophenone ¹H-NMR(300 MHz): δ0.75-0.90(15H, m, Me), 0.95-1.70(24H, br, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂CH₂—O—Ar), 3.82-3.92(2H, br, —O—CH₂ —C₁₉H₃₉), 6.89(1H, d, J = 8.3 Hz,C₃—H), 7.35-7.80(7H, m, C4,6-H, Ph—H)2-chloro-5-(2,3-dihydrophytyloxy)benzhydrol ¹H-NMR(300 M Hz): δ0.82-0.90(15H, m, Me), 1.00-1.90(24H, br, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂CH₂—O—Ar), 3.88-4.00(2H, br, —O—CH₂ —C₁₉H₃₉), 5.98(1H, s, Ar—CHOH—Ph),6.75-6.90(1H, m, C3-H), 7.10-7.45(7H, m, C4,6-H, Ph—H)1-chloro-1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)phenylmethane¹H-NMR(300 MHz): δ 0.80-0.90(15H, m, Me), 1.00-1.90(24H, br, Me₂CH—[C₃H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.88-4.05(2H, m, —O—CH₂ —C₁₉H₃₉), 6.48(1H, d,J = 1.6 Hz, Ar—CHCl—Ph), 6.77(1H, d, J = 8.7 Hz, C3-H), 7.10-7.55(7H, m,C4,6-H, Ph—H)1-azido-1-[(2-chloro-5-(2,3′-dihydrophytyloxy)phenyl)phenylmethane1H-NMR(300 MHz): δ 0.85-0.95(15H, m, Me), 0.95-1.85(24H, br, Me₂CH—[C₃H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.75-4.02(2H, m, —O—CH₂ —C₁₉H₃₉),5.90-6.10(1H, m, Ar—CHN₃—Ph), 6.79(1H, d, J = 9.0 Hz, C3-H),7.10-7.50(7H, m, C4,6-H, Ph—H)1-[(2-chloro-5-(2,3-dihydrophytyloxy)phenyl)]-1-phenylmethanamine¹H-NMR(300 MHz): δ 0.85-0.95(15H, m, Me), 0.95-1.85(24H, br, Me₂CH—[C₃H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.85-4.00(2H, br, —O—CH₂ —C₁₉H₃₉), 5.43(1H, s,Ar—CHNH₂—Ph), 6.75(1H, d, J = 8.7 Hz, C3-H), 7.10-7.50(7H, m, C4,6-H,Ph—H)

TABLE 3 Prep. Ex. 6

¹H-NMR(300 MHz): δ 0.86-0.90(24H, m, Me), 1.10-1.40(48H, br, Me₂CH—[C₃H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 2.03(1H, s, OH), 3.90-3.94(4H, m, —O—CH₂—C₁₉H₃₉), 5.76(1H, s, Ar—CHN₃—Ph), 6.85(4H, m, C3-H), 7.20-7.26(4H, m,C4,6-H, Ph—H) Prep. Ex. 7

¹H-NMR(300 MHz): δ 0.85(36H, t, J = 6.3 Hz, Me), 0.94(9H, t, J = 6.3 Hz,Me), 1.00-2.00(72H, br, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar),3.93-4.07(6H, m, —CH₂ —O—Ar), 4.60(2H, s, —O—CH₂ —OH), 6.57(2H, s,C2,6-H) Prep. Ex. 8

¹H-NMR(300 MHz): δ 0.85(36H, t, J = 6.3 Hz, Me), 0.93(9H, t, J = 6.3 Hz,Me), 1.00-2.00(72H, br, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.79(2H,s, benzyl-H), 3.85-4.10(6H, m, —CH₂ —O—Ar), 6.52(2H, s, C2,6-H) Prep.Ex. 9

¹H-NMR(300 MHz): δ 0.80-0.90(24H, m, Me), 0.93(6H, d, J = 6.3 Hz, Me),1.00-1.90(48 H, br, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.92-4.02(4H,m, C₁₉H₃₉—CH₂ —O—Ar), 4.62(2H, s, Ar—CH₂ —OH), 6.38(1H, t, J = 2.1 Hz,C4-H), 6.50(2H, d, J = 2.0 Hz, C2,6-H) Prep. Ex. 10

4-(2,3-dihydrophytyloxy)benzaldehyde ¹H-NMR(300 MHz): δ 0.82-0.89(12H,m, Me), 0.95(3H, d, J = 6.4 Hz, Me), 1.00-1.95(24 H, m, Me₂CH—[C₃ H₆—CHMe]₃—CH₂ CH₂—O—Ar), 4.03-4.13(2H, m, —O—CH₂ —C₁₉H₃₉), 4.62 (2H, s,Ar—CH₂ —OH), 6.99(2H, m, C3,5-H), 7.83(2H, m, C2,6-H), 9.88(1H, s, CHO)4-(2,3-dihydrophytyloxy)benzyl alcohol ¹H-NMR(300 MHz): δ 0.81-0.90(12H,m, Me), 0.94(3H, d, J = 6.4 Hz, Me), 1.00-1.90(24 H, m, Me₂CH—[C₃ H₆—CHMe]₃—CH₂ CH₂—O—Ar), 3.94-4.05(2H, m, —O—CH₂ —C₁₉H₃₉), 6.89 (2H, m,C3,5-H), 7.28(2H, m, C2,6-H)

TABLE 4 Prep. Ex. 11

4-(2,3-dihydrophytyloxy)benzyl azide ¹H-NMR(300 MHz): δ 0.81-0.90(12H,m, Me), 0.94(3H, d, J = 6.4 Hz, Me), 1.00-1.90(24 H, m, Me₂CH—[C₃ H₆—CHMe]₃—CH₂ CH₂—O—Ar), 3.94-4.04(2H, m, —O—CH₂ —C₁₉H₃₉), 4.26(2H, s,Ar—CH₂ —N₃), 6.90(2H, d, J = 8.6 Hz, C3,5-H), 7.23 (2H, d, J = 8.6 Hz,C2,6-H) 4-(2,3-dihydrophytyloxy)benzylamine ¹H-NMR(300 MHz): δ 0.86(12H,t, J = 6.0 Hz, Me), 0.94(3H, d, J = 6.6 Hz, Me), 1.00- 1.90(24H, m,Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.80(2H, s, Ar—CH₂ —NH₂),3.92-4.04(2H, m, —O—CH₂ —C₁₉H₃₉), 6.87(2H, d, J = 8.6 Hz, C3,5-H), 7.21(2H, d , J = 8.6 Hz, C2,6-H) Prep. Ex. 12

2-methoxy-4-(2,3-dihydrophytyloxy)benzaldoxime ¹H-NMR(300 MHz): δ0.82-0.92(12H, m, Me), 0.95(3H, d, J = 6.4 Hz, Me), 1.00-1.95(24 H, m,Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.83(3H, s, OMe), 3.97-4.10(2H, m,—O—CH₂ —C₁₉H₃₉), 6.44(1H, d, J = 2.2 Hz, C3-H), 6.49(1H, dd, J = 2.2,8.6 Hz, C5-H), 7.15(1H, s, —CHNOH), 7.62(1H, d, J = 8.6 Hz, C6-H),8.41(1H, s, —CHNOH) 2-methoxy-4-(2,3-dihydrophytyloxy)benzylamine¹H-NMR(300 MHz): δ 0.80-0.90(12H, m, Me), 0.94(3H, d, J = 6.4 Hz, Me),1.00-1.90(24 H, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 3.74(2H, s,Ar—CH₂ —NH₂), 3.82(3H, s, OMe), 3.90-4.05(2H, m, —O—CH₂ —C₁₉H₃₉),6.42(1H, dd, J = 2.3, 8.1 Hz, C5-H), 6.46(1H, d, J = 2.1 Hz, C3-H),7.09(1H, d, J = 8.1 Hz, C6-H) Prep. Ex. 13

methyl 4-hydroxy-2-methylbenzoate ¹H-NMR(300 MHz): δ 2.57(3H, s, C2-Me),3.86(3H, s, —COOMe), 5.68(1H, s, br, —OH), 6.66-6.72(2H, m, C3,5-H),7.89(1H, dd, J = 2.4, 6.9 Hz, C6-H) methyl4-(2,3-dihydrophytyloxy)-2-methylbenzoate ¹H-NMR(300 MHz): δ 0.85(12H,t, J = 6.6 Hz, Me), 0.94(3H, d, J = 6.6 Hz, Me), 1.00- 1.90(24H, m,Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 2.59(3H, s, C2-Me), 3.85(3H, s,—COOMe), 4.02(2H, dt, J = 3.0, 6.7 Hz, O—CH₂ —C₁₉H₃₉), 6.65-6.76(2H, m,C3,5-H), 7.85-7.96(1H, m, C6-H) 4-(2,3-dihydrophytyloxy)-2-methylbenzylalcohol ¹H-NMR(300 MHz): δ 0.86(12H, t, J = 6.3 Hz, Me), 0.94(3H, d, J =6.4 Hz, Me), 1.00- 1.90(24H, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar),2.36(3H, s, C2-Me), 3.98(2H, dt, J = 3.0, 6.7 Hz, O—CH₂ —C₁₉H₃₉),4.63(2H, d, J = 4.7 Hz, Ar—CH₂ —OH), 6.60-6.77(2H, m,C3,5-H),7.15-7.25(1H, m, C6-H)

TABLE 5 Prep. Ex. 14

4-(2,3-dihydrophytyloxy)-2-methylbenzyl chloride ¹H-NMR(300 MHz): δ0.86(12H, t, J = 6.3 Hz, Me), 0.93(3H, d, J = 6.3 Hz, Me), 1.00-1.90(24H, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 2.40(3H, s, C2-Me),3.97(2H, dt, J = 2.7, 6.7 Hz, O—CH₂ —C₁₉H₃₉), 4.59(2H, s, Ar—CH₂ —C1),6.69(1H, dd, J = 2.4, 8.3 Hz, C5-H), 6.74(1H, d, J = 2.3 Hz, C3-H),7.21(1H, d, J = 8.3 Hz, C6-H) 4-(2,3-dihydrophytyloxy)-2-methylbenzylazide ¹H-NMR(300 MHz): δ 0.86(12H, t, J = 6.3 Hz, Me), 0.94(3H, d, J =6.3 Hz, Me), 1.00-1.90 (24H, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar),2.34(3H, s, C2-Me), 3.98(2H, dt, J = 3.0, 6.6 Hz, O—CH₂ —C₁₉H₃₉),4.28(2H, s, Ar—CH₂—N3), 6.71(1H, dd, J = 2.6, 8.2 Hz, C5-H), 6.77(1H, d,J = 2.3 Hz, C3-H), 7.15(1H, d, J = 8.3 Hz, C6-H)4-(2,3-dihydrophytyloxy)-2-methylbenzylamine ¹H-NMR(300 MHz): δ0.86(12H, t, J = 6.3 Hz, Me), 0.93(3H, d, J = 6.6 Hz, Me), 1.00-1.90(24H, m, Me₂CH—[C₃ H₆ —CHMe]₃—CH₂ CH₂—O—Ar), 2.32(3H, s, C2-Me),3.79(2H, s, Ar—CH₂ —NH₂), 3.97(2H, dt, J = 3.0, 6.7 Hz, O—CH₂ —C₁₉H₃₉),6.71(1H, dd, J = 2.6, 8.2 Hz, C5-H), 6.68-6.75(2H, br, C3,5-H), 7.17(1H,d, J = 8.7 Hz, C6-H) Prep. Ex. 15

¹H-NMR(300 MHz): δ 0.92-0.99(m, 24H), 1.01-1.09(m, 6H), 1.19-1.23(m,4H), 4.58(s, 2H), 7.21(d, 2H, J = 6 Hz), 7.43(d, 2H, J = 9 Hz),7.53-7.66(b, 1H) Prep. Ex. 16

¹H-NMR(300 MHz): δ 0.81-0.89(m, 12H), 1.08-1.37(m, 12H), 1.48-1.83(m,5H), 3.96- 4.02(m, 2H), 4.62(s, 2H), 6.89(d, 2H, J = 9 Hz), 7.29(d, 2H,J = 9 Hz) Prep. Ex. 17

¹H-NMR(400 MHz, CDCl₃): δ 0.84(d, 6H, J = 6.6 Hz), 0.87(d, 6H, J = 6.6Hz), 0.92(d, 3H, J = 6.6 Hz), 1.00-1.44(m, 21H), 1.47-1.58(m, 1H),1.91-1.99(m, 1H), 2.15(ddd, 1H, J = 15.0, 8.2, 2.0 Hz), 2.36(ddd, 1H, J= 15.0, 5.9, 1.9 Hz)

TABLE 6 Prep. Ex. 18

¹H-NMR(400 MHz, CDCl₃): δ 0.84(d, 6H, J = 6.6 Hz), 0.87(d, 6H, J = 6.6Hz), 0.92(d, 3H, J = 6.6 Hz), 1.00-1.44(m, 21H), 1.47-1.58(m, 1H),1.91-1.99(m, 1H), 2.15(ddd, 1H, J = 15.0, 8.2, 2.0 Hz), 2.36(ddd, 1H, J= 15.0, 5.9, 1.9 Hz) Prep. Ex. 19

¹H-NMR(400 MHz, CDCl₃): δ 0.84(d, 6H, J = 6.6 Hz), 0.87(d, 6H, J = 6.6Hz), 0.96(d, 3H, J = 6.6 Hz), 1.01-1.42(m, 20H), 1.45-1.57(m, 1H),1.93-2.02(m, 1H), 2.18(ddd, 1H, J = 14.9, 8.1, 1.8 Hz), 2.37(ddd, 1H, J= 14.9, 5.9, 1.8 Hz), 5.70(d, 1H, J = 6.0 Hz), 5.71(d, 1H, J = 6.0 Hz)Prep. Ex. 20

¹H-NMR(400 MHz, CDCl₃): δ 0.87(d, 6H, J = 6.6 Hz), 0.95(d, 3H, J = 6.5Hz), 1.12- 1.38(m, 6H), 1.50-1.63(m, 3H), 1.82-1.90 (m, 1H),4.04-4.11(m, 1H), 6.93(d, 2H, J = 8.8 Hz), 8.06(d, 2H, J = 8.8 Hz) Prep.Ex. 21

¹H-NMR(300 MHz, CDCl₃): δ 0.99-1.16 (m, 12H), 2.14-2.35(m, 1H), 2.42(t,1H, J = 6.5 Hz), 2.55-2.74(m, 2H), 3.30-3.40 (m, 1H), 3.45-3.66(m, 4H),3.70-3.87(m, 7H), 4.10-4.14(m, 1H), 4.52-4.69(m, 1H), 5.35(d, 1H, J =7.2 Hz), 6.27-6.38(m, 1H), 6.79-6.88(m, 4H), 7.20-7.35(m, 7H), 7.35-7.44(m, 2H), 7.94, 8.03(2d, 1H, J = 7.4 Hz) ³¹P-NMR(120 MHz, CDCl₃): δ149.7, 149.1 Prep. Ex. 22

¹H-NMR(400 MHz, CDCl₃): δ 1 .04-1.19 (m, 12H), 2.44-2.61(m, 3H),2.72-2.85(m, 1H), 3.29-3.40(m, 2H), 3.53-3.90(m, 10H), 4.24(s, 1H),4.70-4.73(m, 1H), 6.02(brs, 2H), 6.21(t, 1H, J = 7.02 Hz), 6.78(dd, 4H,J = 2.70, 8.64 Hz), 7.14-7.30(m, 7H), 7.38- 7.41(m, 2H), 7.65(d, 1H, J =4.05 Hz) ³¹P-NMR(160 MHz, CDCl₃): δ 149.5, 149.3 Prep. Ex. 23

TABLE 7 Prep. Ex. 24

2,3,4-tris(2,3-dihydrophytyloxy)benzophenone ¹H-NMR(400 MHz, CDCl₃): δ0.68-1.95(m, 117H), 3.88-3.95(m, 2H), 3.97-4.11(m, 4H), 6.71(d, 1H, J =8.7 Hz), 7.14(d, 1H, J = 8.6 Hz), 7.38-7.44(m, 2H), 7.50-7.55(m, 1H),7.76- 7.79(m, 2H) 2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylalcohol¹H-NMR(400 MHz, CDCl₃): δ 0.78-1.95(m, 117H), 3.05-3.10(m, 1H),3.68-3.78(m, 1H), 3.93-4.05(m, 5H), 5.89-5.94(m, 1H), 6.61(d, 1H, J =8.6 Hz), 6.82-6.86(m, 1H), 7.20-7.28 (m, 1H), 7.29-7.34(m, 2H),7.35-7.39(m, 2H)N-(9-fluorenylmethoxycarbonyl)-2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylamine¹H-NMR(400 MHz, CDCl₃): δ 0.83-1.90(m, 117H), 3.37-3.45(m, 1H),3.92-4.06(m, 5H), 4.22-4.28(m, 1H), 4.35-4.42(m, 1H), 4.42-4.50(m, 1H),5.90-5.98(m, 1H), 6.01-6.08(m, 1H), 6.63(d, 1H, J = 8.5 Hz),6.89-6.94(m, 1H), 7.14-7.43(m, 9H), 7.58-7.65(m, 2H), 7.73- 7.78(m, 2H)2,3,4-tris(2,3-dihydrophytyloxy)benzhydrylamine ¹H-NMR(400 MHz, CDCl₃):δ 0.83-1.90(m, 117H), 3.77-3.91(m, 2H), 3.91-4.02(m, 4H), 5.40(s, 1H),6.60(d, 1H, J = 8.6 Hz), 6.90(d, 1H, J = 8.4 Hz), 7.20-7.37(m, 5H) Prep.Ex. 25

N-(9-fluorenylmethoxycarbonyl)-bis-4-(2,3-dihydrophytyloxy)benzhydrylamine¹H-NMR(400 MHz, CDCl₃): δ 0.83-1.85(m, 78H), 3.90-4.02(m, 4H),4.18-4.25(m, 1H), 4.35-4.44(m, 2H), 5.20-5.30(m, 1H), 5.83-5.91(m, 1H),6.84(d, 4H, J = 8.9 Hz), 7.11(d, 4H, J = 8.6 Hz), 7.24-7.43(m, 4H),7.53-7.62(m, 2H), 7.70-7.78(m, 2H)4,4′-bis(2,3-dihydrophytyloxy)benzhydrylamine ¹H-NMR(400 MHz, CDCl₃) δ0.80-0.93(m, 30H), 1.01-1.85(m, 52H), 3.93-3.98(m, 4H), 5.12(s, 1H),6.83(d, 4H, J = 8.7 Hz), 7.24(d, 4H, J = 8.9 Hz) Prep. Ex. 26

3,5-bis(2,3-dihydrophytyloxy)benzyl chloride ¹H-NMR(400 MHz, CDCl₃): δ0.83-1.85(m, 78H), 3.90-4.02(m, 4H), 4.50(s, 2H), 6.40(d, 1H, J = 2.2Hz), 6.51(d, 2H, J = 2.2 Hz) 3,5-bis(2,3-dihydrophytyloxy)benzyl azide¹H-NMR(400 MHz, CDCl₃): δ 0.83-1.85(m, 78H), 3.90-4.02(m, 4H), 4.50(s,2H), 6.40- 6.45(m, 3H) 3,5-bis(2,3-dihydrophytyloxy)benzyl amine¹H-NMR(400 MHz, CDCl₃): δ 0.82-1.88(m, 78H), 3.79(s, 2H), 3.92-4.01(m,4H), 6.34(t, 1H, J = 2.2 Hz), 6.45(d, 2H , J = 2.2 Hz)

TABLE 8 Prep. Ex. 27

5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine ¹H-NMR(400 MHz,CDCl₃): δ 3.50-3.64(m, 2H), 3.80(s, 6H), 4.08(m, 1H), 4.49-4.60(m, 1H),5.03(dd, 1H, J = 4.2, 51.2 Hz), 5.33(d, 1H, J = 8.2 Hz), 6.07(d, 1H, J =14.9 Hz), 6.83-7.40(m, 13H), 7.90(d, 1H, J = 8.2 Hz) 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-2′-fluorouridine-3′-O-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite] ¹H-NMR(400 MHz, CDCl₃): δ 0.99-1.32(m, 12H),2.40-2.65(m, 2H), 3.40-3.93(m, 6H), 3.80(m, 6H), 4.20-4.30(m, 1H),4.55-4.80(m, 1H), 5.00-5.23(m, 1H), 5.24-5.29(m, 1H), 6.07(d, 2H, J =16.1 Hz), 6.82-7.44(m, 13H), 7.92-8.04(m, 1H) ³¹P-NMR(160 MHz, CDCl₃): δ149.7, 150.3 Ex. 1

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.97(m, 45H), 0.98-(m, 75H), 2.38-2.48(m,2H), 2.60- 2.71(m, 4H), 3.39-3.51(m, 2H), 3.79(s, 6H), 3.85-4.20(m, 6H),4.13-4.15(m, 1H), 5.40- 5.50(m, 1H), 6.38-6.45(m, 1H), 6.54(s, 2H),6.84(d, 4H, J = 8.9 Hz), 7.20-7.39(m, 9H), 7.60(s, 1H) Ex. 2

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.97(m, 45H), 1.00-2.00(m, 75H),2.42-2.48(m, 2H), 2.52(t, 2H, J = 6.6 Hz), 2.71(t, 2H, J = 6.6 Hz),3.79(s, 6H), 3.47(ddt, 2H, J = 2.4, 5.6, 16.2 Hz), 3.91-4.00(m, 6H),4.13-4.17(m, 1H), 5.46-5.50(m, 1H), 6.40-6.45(m, 1H), 6.47(s, 2H),6.82-6.85(m, 4H), 7.22-7.39(m, 9H), 7.61(d, 1H, J = 1.2 Hz)

TABLE 9 Ex. 4

¹H-NMR(400 MHz, CDCl₃): δ 0.87(d, 6H, J = 6.6 Hz), 0.93(d, 3H, J = 6.4Hz), 1.10-1.89(m, 10H), 1.93(s, 3H), 2.25-2.40(m, 2H), 2.81(brs, 1H),3.48(dd, 1H, J = 7.1, 14.0 Hz), 3.72- 3.91(m, 2H), 3.92(d, 1H, J = 2.9Hz), 4.00-4.12(m, 2H), 4.49(m, 1H), 6.19(m, 1H), 6.93(d, 2H, J = 8.7Hz), 7.60(s, 1H), 7.86(d, 2H, J = 8.7 Hz) Ex. 5

¹H-NMR(400 MHz, CDCl₃): δ 0.87(d, 6H, J = 6.6), 0.93(d, 3H, J = 6.5 Hz),1.12-1.88(m, 13H), 2.38-2.46(m, 2H), 3.39(dd, 1H, J = 3.1, 10.6 Hz),3.51(dd, 1H, J = 3.1, 10.6 Hz), 3.80(s, 6H), 4.01-4.10(m, 3H),4.57-4.62(m, 1H), 6.39(t, 1H, J = 6.6 Hz), 6.83-6.88(m, 4H), 6.92(d, 2H,J = 9.0 Hz), 7.24-7.42(m, 9H), 7.68(s, 1H), 7.88(d, 2H, J = 9.0 Hz) Ex.6

¹H-NMR(400 MHz, CDCl₃): δ 0.87(d, 6H, J = 6.6 Hz), 0.93(d, 3H, J = 6.4Hz), 1.14-1.17(m, 12H), 1.17-1.90(m, 13H), 2.32-2.43(m, 1H),2.45-2.55(m, 1H), 2.43(t, 2H, J = 6.3 Hz), 2.60 (t, 2H, J = 6.3 Hz),3.32-3.38(m, 1H), 3.49-3.85(m, 5H), 3.80, 3.81(2s, 6H), 4.02-4.09 (m,2H), 4.13, 4.19(2m, 1H), 4.63-4.71(m, 1H), 6.36-6.42(m, 1H),6.84-6.87(m, 4H), 6.93 (d, 2H, J = 8.8 Hz), 7.13-7.19(m, 2H),7.22-7.35(m, 5H), 7.39-7.44(m, 2H), 7.70, 7.75(2s, 1H), 7.89(d, 2H, J =8.8 Hz), 7.90(d, 2H, J = 8.8 Hz) ³¹P-NMR(400 MHz, CDCl₃): δ 149.7, 150.3Ex. 7

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.87(m, 12H), 0.94(d, 3H, J = 6.6 Hz),1.04-1.58(m, 21H), 1.89-2.01(m, 1H), 2.11(ddd, 1H, J = 2.6, 8.5, 14.9Hz), 2.19(s, 3H), 2.31-2.37(m, 1H), 2.42-2.51(m, 2H), 2.57(t, 2H, J =6.2 Hz), 2.75(dt, 2H, J = 2.4, 6.4 Hz), 3.45(dd, 1H, J = 2.4, 10.5 Hz),3.48(dd, 1H, J = 2.4, 10.5 Hz), 3.79(s, 6H), 4.12-4.15(m, 1H),5.46-5.50(m, 1H), 5.98(t, 2H, J = 7.3 Hz), 6.46(dd, 1H, J = 5.7, 8.8Hz), 6.81-6.85(m, 4H), 7.14-7.39(m, 9H), 7.64(d, 1H, J = 1.1 Hz)

TABLE 10 Ex. 8

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.87(m, 12H), 0.93(d, 3H, J = 6.6 Hz),1.02-1.56(m, 24H), 1.88-1.99(m, 1H), 2.10(ddd, 1H, J = 3.0, 6.9, 15.9Hz), 2.31-2.36(m, 2H), 2.42(ddd, 1H, J = 3.0, 6.9, 15.9 Hz), 3.38(dd,1H, J = 3.0, 10.5 Hz), 3.49(dd, 1H, J = 3.0, 10.5 Hz), 3.75- 3.81(m,2H), 3.79(s, 6H), 4.04(dt, 1H, J = 3.1, 6.2 Hz), 4.56-4.60(m, 1H),5.98(dt, 2H, J = 1.9, 11.2 Hz), 6.42(dt, 1H, J = 1.2, 7.3 Hz),6.72-6.78(m, 1H), 6.82-6.86(m, 4H), 7.10-7.41 (m, 9H), 7.60(d, 1H, J =1.2 Hz) Ex. 9

m/z(ESI-MS): Anal. Calc. for C₆₁H₈₉N₄O₁₀P: 1068.6. Found 1069.4(M + H)⁺Ex. 10

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.87(m, 12H), 0.94(d, 3H, J = 6.6 Hz),1.03-1.58(m, 37H), 1.94-1.97(m, 1H), 2.07-2.14(m, 1H), 2.29-2.37(m, 1H),2.41(t, 1H, J = 6.3 Hz), 2.46- 2.59(m, 1H), 2.61(t, 1H, J = 6.3 Hz),3.31-3.35(m, 1H), 3.47-3.64(m, 4H), 3.74-3.85(m, 1H), 3.78, 3.79(2s,6H), 4.14, 4.18(2d, 1H, J = 2.2 Hz), 4.63-4.69(m, 1H), 5.98(s, 2H),6.40-6.46(m, 1H), 6.82-6.86(m, 4H), 7.15-7.18(m, 2H), 7.23-7.32(m, 5H),7.39-7.41(m, 2 H), 7.63, 7.68(2d, 1H, J = 1.1 Hz) ³¹P-NMR(400 MHz,CDCl₃): δ 149.7, 150.3 Ex. 11

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.87(m, 27H), 1.09-1.52(m, 22H),2.08-2.19(m, 1H), 2.43-2.49(m, 2H), 2.62(t, 1H, J = 6.3 Hz),2.70-2.76(m, 1H), 2.86-2.95(m, 1H), 3.29-3.88 (m, 6H), 3.77(s, 6H),4.26-4.30(m, 1H), 4.73-4.76(m, 1H), 6.27-6.30(m, 1H), 6.75-7.42 (m,13H), 7.85, 7.86(2S, 1H) ³¹P-NMR(400 MHz, CDCl₃): δ 149.9, 150.0m/z(ESI-MS): Anal. Calc. for C₆₀H₈₆N₇O₈P: 1063.6. Found 1062.4(M − H)⁻

TABLE 11 Ex. 12

¹H-NMR(400 MHz, CDCl₃): δ 0.82-0.87(m, 24H), 0.93(d, 3H, J = 6.6 Hz),1.02-1.55(m, 22H), 1.87-1.93(m, 1H), 1.99-2.02(m, 1H), 2.22-2.26(m, 1H),2.38-2.49(m, 1H), 2.45(t, 1H, J = 6.5 Hz), 2.60(t, 1H, J = 6.5 Hz),3.31-3.42(m, 2H), 3.57-3.63(m, 2H), 3.66-3.86(m, 2H), 3.76, 3.77(2s,6H), 4.26-4.33(m, 1H), 4.70-4.78(m, 1H), 6.41-6.49(m, 1H), 6.76-6.81 (m,4H), 7.15-7.48(m, 9H), 7.97, 7.99(2s, 1H), 8.61, 8.85(2s, 1H)³¹P-NMR(400 MHz, CDCl₃): δ 149.9, 150.0 m/z(ESI-MS): Anal. Calc. forC₆₀H₈₆N₇O₇P: 1047.6. Found 1046.3(M − H)⁻ Ex. 19

¹H-NMR(300 MHz, CDCl₃): δ 0.83-0.95 (m, 6H), 1.03-1.35(m, 16H),1.42-1.73 (m, 4H), 2.10-2.33(m, 2H), 2.44(t, 1H, J = 6.5 Hz), 2.62(t,1H, J = 6.3 Hz), 2.68-2.85 (m, 1H), 3.30-3.90(m, 6H), 3.81(s, 6H),4.20-4.24(m, 1H), 4.53-4.65(m, 1H), 6.18-6.28(m, 1H), 6.82-6.89(m, 4H),7.13-7.44(m, 9H), 7.79(brs, 1H), 8.19, 8.29(2d, 1H, J = 7.4 Hz)³¹P-NMR(120 MHz, CDCl₃): δ 149.9, 149.2 Ex. 20

¹H-NMR(400 MHz, CDCl₃): δ 0.90(s, 9H), 0.99-1.30(m, 18H), 2.03-2.83(m,6H), 3.33-3.88(m, 12H), 3.34-3.67(m, 5H), 3.69-3.86(m, 1H),4.18-4.4.24(m, 1H), 4.54-4.68(m, 1H), 6.22-6.29(m, 1H), 6.82-6.88(m,4H), 7.10-7.43(m, 18H), 8.17-8.29(m, 2H) ³¹P-NMR(160 MHz, CDCl₃): δ150.6, 150.0 Ex. 21

¹H-NMR(300 MHz, CDCl₃): δ 0.78-1.50 (m, 46H), 2.13-2.85(m, 5H),3.34-3.95 (m, 12H), 4.20-4.24(m, 1H), 4.53-4.68 (m, 1H), 6.22-6.31(m,1H), 6.78-6.92 (m, 4H), 7.10-7.45(m, 9H), 7.80-7.89 (m, 1H),8.12-8.32(m, 1H) ³¹P-NMR(120 MHz, CDCl₃): δ 149.8, 149.1

TABLE 12 Ex. 22

¹H-NMR(400 MHz, CDCl₃): δ 0.84-0.92(m, 6H), 1.05- 1.20(m, 12H),1.21-1.75(m, 28H),2.07-2.85(m, 5H), 3.34-3.88(m, 6H), 3.80, 3.81(2s,6H), 4.20-4.24 (m, 1H), 4.53-4.66(m, 1H), 6.23-6.30(m, 1H), 6.82-6.88(m, 4H), 7.12-7.42(m, 10H), 7.89-7.99(m, 1H), 8.18, 8.27(2d, 1H, J = 7.4Hz) ³¹P-NMR(160 MHz, CDCl₃): δ 150.0, 150.6 Ex. 23

¹H-NMR(300 MHz, CDCl₃): δ 0.81-0.93(m, 6H), 1.05- 1.20(m, 12H),1.20-1.80(m, 24H), 2.18-2.85(m, 5H), 3.32-3.88(m, 6H), 3.80, 3.81(2s,6H), 4.19-4.24 (m, 1H), 4.53-4.68(m, 1H), 6.22-6.31(m, 1H), 6.81-6.88(m, 4H), 7.13-7.45(m, 10H), 8.02-8.12(m, 1H), 8.19, 8.29(2d, 1H, J = 7.5Hz) ³¹P-NMR(120 MHz, CDCl₃): δ 149.2, 149.8 Ex. 24

¹H-NMR(400 MHz, CDCl₃): δ 0.84-0.92(m, 3H), 1.03- 1.20(m,12H),1.20-1.78(m, 22H), 2.23-2.85(m, 6H), 3.34-3.87(m, 6H), 3.80, 3.81(2s, 6H), 4.20-4.24(m, 1H), 4.55-4.66(m, 1H), 6.22-6.30 (m, 1H),6.80-6.87(m, 4H), 7.08-7.42(m, 9H), 7.83 (brs, 1H), 8.19, 8.29 (2d, 1H,J = 7.5 Hz) ³¹P-NMR(160 MHz, CDCl₃): δ 150.0, 150.5 Ex. 25

¹H-NMR(400 MHz, CDCl₃): δ 0.84-0.91(m, 12H), 0.92- 0.98(m,6H),1.07-1.72(m, 28H), 2.45-2.65(m, 4H), 3.27-3.95(m, 12H), 4.22-4.28(m, 1H),4.63-4.72 (m, 1H), 6.36-6.45(m, 1H), 6.77-6.85(m,4H),7.15-7.34 (m, 13H), 7.71-7.74(m, 1H), 8.41(brs, 1H), 8.58-8.64 (m,1H) ³¹P-NMR(160 MHz, CDCl₃): δ 149.9, 150.1 Ex. 33

¹H-NMR(400 MHz, CDCl₃): δ 0.67-1.93(m, 120H), 2.40- 2.83(m, 6H),3.20-3.33(m, 1H), 3.46(m, 2H), 3.78(s, 6H), 3.98(m, 5H), 4.13 (brs, 1H),5.50(m, 1H), 6.23-6.28(m, 1H), 6.40-6.46 (m, 1H), 6.59-6.63 (m, 1H),6.74-7.43(m, 20H), 7.58- 7.63(m, 1H), 8.28(brs, 1H)

TABLE 13 Ex. 34

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.95 (m, 30H), 1.00-1.85(m, 51H),2.40-2.45 (m, 2H), 2.52-2.58(m, 2H), 2.67-2.74 (m, 2H), 3.45(d, 2H, J =2.3 Hz), 3.78(s, 6H), 3.85-3.97(m, 4H), 4.10(brs, 1H), 5.48 (m, 1H),6.09(dd, 2H, J = 7.9, 13.6 Hz), 6.40(m, 1H), 6.78-7.41(m, 21H), 7.60 (m,1H), 8.14(brs, 1H) Ex. 35

¹H-NMR(400 MHz, CDCl₃): δ 0.83-0.95 (m, 30H), 1.00-1.85(m, 51H), 2.40-2.55 (m, 4H), 2.67-2.74(m, 2H), 3.43-3.50 (m, 1H), 3.78(s, 6H),3.88-3.97(m, 4H), 4.14(m, 1H), 4.35(d, 1H, J = 5.5 Hz), 5.45-5.50(m,1H), 5.72-5.77(m, 1H), 6.35-6.45(m, 4H), 6.80-7.40(m, 13H), 7.60(m, 1H),7.94(brs, 1H) Ex. 43

1H-NMR(400 MHz, CDCl₃): δ 0.83-0.95(m, 15H), 0.97-1.93(m, 39H),2.35-2.40(m, 1H), 2.52-2.63(m, 1H), 3.37-3.60(m, 6H), 3.78-3.90(m, 8H),4.03-4.13(m, 2H), 4.33-4.42 (m, 1H), 4.59(d, 1H, J = 26.5 Hz), 5.67(d,1H, J = 2.2 Hz), 6.81-6.88(m, 4H), 6.90-6.95(m, 2H), 4.01-4.12(m, 2H),6.91-6.96(m, 2H), 7.23-7.36(m, 7H), 7.42-7.49(m, 2H), 7.68-7.74 (m, 1H),8.03-8.08(m, 2H) Comp. TLC: Rf = 0.50(dichloromethane:methanol = 4:1 Ex.2 ¹H-NMR(400 MHz): δ 0.89(t, 9H, J = 7.0 Hz, H3C(octadecyloxy)),1.25-1.79(m, 102H, —CH2-(octadecyloxy)), 1.35(s, 3H, N5—CH3-thymidine),2.45(m, 2H, 2′-thymidine), 2.51(m, 2H, succinyl), 2.70(m, 2H, succinyl),3.46(m, 2H, 5′-thymidine), 3.79(s, 6H, H3CO-DMTr), 3.79-3.95(m, 6H,Bn-O—CH2—), 4.15(m, 1H, 4′-thymidine), 4.32(d, 2H, J = 5.5 Hz,—NH—CH2-benzyl), 5.47(m, 1H, 3′-thymidine), 5.72(d, 2H, J = 5.5 Hz,—NH—CH2-benzyl), 6.41(m, 1H, 1′-Thymidine), 6.45(s, 2H, -benzyl),6.83(d, 4H, J = 9.0 Hz, DMTr), 7.24-7.38 (m, 9H, DMTr), 7.61(s, 1H,N6-thymidine), 7.95(br•s, N3—NH-thymidine)

Experimental Example 1 Solubility Test of Nucleoside with Protected3′-hydroxyl Group

The solubility (=solute/(solvent+solute)×100) (mass %) at 20° C., shownin the following Table 2, of the compound representing the presentinvention, thymidine wherein the 5′-hydroxyl group is protected bydimethoxytrityl group, and the 3′-hydroxyl group is protected by thebranched chain-containing aromatic protecting group (Example 2), andthymidine wherein the 5′-hydroxyl group is protected by dimethoxytritylgroup, and the 3′-hydroxyl group is protected by the corresponding groupcontaining the straight chain structure (Comparative Example 2) as acomparison target thereof was measured.

[Solubility Measurement Method]

-   1) A solute (100 parts by mass) was added to a solvent (100 parts by    mass) to saturation at 20° C.-   2) When the solute remained by visual observation, the supernatant    was quantitatively analyzed under the following HPLC conditions, and    the concentration was determined and taken as the solubility    (=solute/(solvent+solute)×100)(mass %).-   3) When the solute was absent by visual observation, the solubility    was >50 mass %.    [HPLC Analysis Conditions]-   use instrument: Hitachi high performance liquid chromatography    LaChrom Elite L-2000 series-   column: YMC-PACK 5 μm 150×4.6 mm-   column temperature: 40° C.-   eluent: THF/CH₃CN/H₂O-   flow rate: 1.0 ml/min

TABLE 14 Example 2 Comparative Example 2 Solvent

heptane >50% by mass   0% by mass heptane/ >50% by mass 22.3% by masstoluene = 1/1 heptane/ >50% by mass 13.7% by mass toluene/ acetonitrile= 1/1/1

The nucleoside protected by a branched chain-containing aromatic groupwherein the nucleoside 3′-hydroxyl group is protected by a branchedchain-containing aromatic protecting group of the present invention wasfound to show a remarkable solubility in heptane (the mostrepresentative non-polar solvent) which is preferably used for reactionsolvents and extraction solvents in the present invention, and a solventof an appropriate mixture of heptane with toluene (non-polar solventwith different polarity from heptane) or acetonitrile (polar solvent),as compared to a nucleoside protected by a group having a straight chainstructure similar to that of a known nucleotide 3′-hydroxyl-protectinggroup (described in JP-A-2010-275254).

Industrial Applicability

Using the particular oligonucleotide comprising a protected base of thepresent invention, a production method of oligonucleotide by aphosphoramidite method wherein the oligonucleotide can be purified by aliquid-liquid extraction operation efficiently and in a high yield canbe provided.

Using the particular oligonucleotide comprising a protected base of thepresent invention, liposolubility and solubility in an organic solvent(particularly, non-polar solvent) of an intermediate oligonucleotideobtained in each step of a nucleotide elongation reaction are strikinglyimproved to enable isolation and purification by an extraction operationalone, and therefore, a complicated, time-consuming operation such assolidification isolation and the like is not required, the speedincreases, and the efficiency and producibility of synthesis ofoligonucleotide with high polymerization degree is strikingly improved.

Furthermore, by using the (oligo)nucleotide comprising a protected baseof the present invention, which is imparted with liposoluble andsolubility in organic solvents (particularly, non-polar solvents),adding a particular cation scavenger during or after deprotection of the5′-terminal hydroxyl group protected by a temporary protecting group,applying a neutralization treatment after completion of the deprotectionreaction, and using a particular oxidizing agent or sulfurizing agent inan oxidation step or a sulfurization step, for a nucleotide elongationreaction including (1) a deprotection step of 5′-terminal hydroxyl groupprotected by a temporary protecting group, (2) an elongation step of5′-terminal by the addition of an oligonucleotide comprising a protectedbase, and (3) a phosphite triester moiety oxidizing step or sulfurizingstep, steps (1), (2) and (3) can be performed in a liquid andoligonucleotide, which is an elongated nucleotide, can be isolated andpurified by an extraction operation alone, and therefore, the elongationreaction in the next cycle can be sequentially performed without takingout the resultant product from the reaction apparatus, wherebyoligonucleotide can be produced continuously in one pot.

In addition, since in the production method of oligonucleotide of thepresent invention, the oligonucleotide can be stably dissolved in ortransferred to a non-polar solvent irrespective of the sequence andchain length of oligonucleotide even as compared to conventional liquidphase methods, it is advantageous in that the isolation and purificationstep can be simplified as for the steps, and high purity and high yieldcan be ensured as a total view.

This application is based on a patent application Nos. 2012-033429 and2012-254718 filed in Japan, the contents of which are incorporated infull herein.

The invention claimed is:
 1. A compound comprising a protected base,which is represented by the formula (I):

wherein q is any integer of not less than 0; Base² in the number of q+1are each independently a nucleic acid base protected by a group having aC₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀straight chain or branched chain alkenyl group; P¹ is a hydrogen atom,or a temporary protecting group removable under acidic conditions; X isa hydrogen atom, an optionally protected hydroxyl group, a halogen atomor an organic group crosslinked with the 4-position carbon atom; X′ inthe number of q are each independently a hydrogen atom, an optionallyprotected hydroxyl group, a halogen atom or an organic group crosslinkedwith the 4-position carbon atom; P² in the number of q+1 are eachindependently a protecting group removable under basic conditions; R³⁴in the number of q are each independently an oxygen atom or a sulfuratom; and R_(e) and R_(f) are each independently a C₁₋₆ alkyl group, ora 5- or 6-membered saturated cyclic amino group formed together with theadjacent nitrogen atom.
 2. The compound comprising a protected base ofaccording to claim 1, wherein q is
 0. 3. The compound comprising aprotected base according to claim 1 or 2, wherein the group having aC₅₋₃₀ straight chain or branched chain alkyl group and/or a C₅₋₃₀straight chain or branched chain alkenyl group is a group represented bythe formula (k):

wherein * indicates the bonding position to a nucleic acid base; R²⁷ isa C₅₋₃₀ straight chain or branched chain alkyl group or a C₅₋₃₀ straightchain or branched chain alkenyl group; a group represented by theformula (1):

wherein * indicates the bonding position to a nucleic acid base; Q₁ is—O—, —S—or —NR³⁰— wherein R³⁰ is a hydrogen atom or a C₁₋₂₂ alkyl group;R_(c) and R_(d) are each independently a hydrogen atom or a C₁₋₂₂ alkylgroup; and R²⁸ is a C₅₋₃₀ straight chain or branched chain alkyl groupor a C₅₋₃₀ straight chain or branched chain alkenyl group; a grouprepresented by the formula (m):

wherein * indicates the bonding position to a nucleic acid base; 1 is aninteger of 1 to 5; Q₂ in the number of 1 are each independently a singlebond, or —O—, —S—, —OC(═O)—, —C(═O)O—, —O—CH₂—, —NH—, —NHC(═O)—,—C(═O)NH—, —NH—CH₂— or —CH₂—; R²⁹ in the number of 1 are eachindependently a C₅₋₃₀ straight chain or branched chain alkyl group or aC₅₋₃₀ straight chain or branched chain alkenyl group; ring C is abenzene ring or a cyclohexane ring, each optionally having, in additionto Q₂R²⁹ in the number of 1 and *C═O, a substituent selected from thegroup consisting of a halogen atom, a C₁₋₆ alkyl group optionallysubstituted by one or more halogen atoms, and a C₁₋₆ alkoxy groupoptionally substituted by one or more halogen atoms; or a grouprepresented by the formula (s):

wherein * indicates the position at which an imino bond is formed withan amino group of a nucleic acid base; and R³⁵ and R³⁶ are eachindependently a C₅₋₃₀ straight chain or branched chain alkyl group or aC₅₋₃₀ straight chain or branched chain alkenyl group.
 4. The compoundcomprising a protected base according to claim 3, wherein R²⁷, R²⁸, R²⁹in the number of 1, R³⁵ and R³⁶ are each independently a branched chainalkyl group or branched chain alkenyl group selected from the groupconsisting of a 2,6,10,14-tetramethylpentadecyl group, a2,6,10-trimethylundecyl group, a 2,2,4,8,10,10-hexamethyl-5-undecylgroup, a 2,6,10-trimethylundeca-1,5,9-trienyl group, a2,6-dimethylheptyl group, a 2,6-dimethylhept-5-enyl group, a2,6-dimethylhepta-1,5-dienyl group, a 9-nonadecyl group, a12-methyltridecyl group, an 11-methyltridecyl group, an 11-methyldodecylgroup, a 10-methylundecyl group, an 8-heptadecyl group, a 7-pentadecylgroup, a 7-methyloctyl group, a 3-methyloctyl group, a 3,7-dimethyloctylgroup, a 3-methylheptyl group, a 3-ethylheptyl group, a 5-undecyl group,a 2-heptyl group, a 2-methyl-2-hexyl group, a 2-hexyl group, a 3-heptylgroup, a 4-heptyl group, a 4-methyl-pentyl group, a 3-methyl-pentylgroup, and a 2,4,4-trimethylpentyl group; or a straight chain alkylgroup selected from the group consisting of a tetradecyl group, atridecyl group, a dodecyl group, an undecyl group, a decyl group, anonyl group, an octyl group, a heptyl group, a hexyl group, and a pentylgroup.
 5. The compound comprising a protected base according to claim 1,wherein the C₅₋₃₀ straight chain or branched chain alkyl group and/orC₅₋₃₀ straight chain or branched chain alkenyl group is a C₅₋₃₀ branchedchain alkyl group and/or a C₅₋₃₀ branched chain alkenyl group.
 6. Thecompound comprising a protected base according to claim 1, wherein P¹isa monomethoxytrityl group or a dimethoxytrityl group.
 7. A method ofproducing an n+p-mer oligonucleotide, comprising (2) a step ofcondensing a p-mer oligonucleotide comprising a protected base, whereinp is any integer of one or more, wherein the 3′-hydroxyl group isphosphoramidited, the 5′-hydroxyl group is protected by a temporaryprotecting group removable under acidic conditions, and the nucleic acidbase is protected by a group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ straight chain or branched chainalkenyl group, with an n-mer oligonucleotide, wherein n is an integer ofone or more, wherein the 5′-hydroxyl group is not protected and the3′-hydroxyl group is protected, by forming a phosphite triester bond viathe 5′-hydroxyl group thereof in a liquid phase.
 8. The productionmethod according to claim 7, wherein p is
 1. 9. The production methodaccording to claim 7 or 8, further comprising the following step (3):(3) a step of converting the phosphite triester bond of the n+p-meroligonucleotide obtained in the condensation step to a phosphatetriester bond or a thiophosphate triester bond by adding an oxidizingagent or a sulfurizing agent to the reaction mixture obtained in thecondensation step (2).
 10. The production method according to claim 7,further comprising the following step (1): (1) a step of removing thetemporary protecting group removable under acidic conditions of the5′-hydroxyl group by reacting, in a non-polar solvent prior to thecondensation step (2), an n-mer oligonucleotide wherein the 3′-hydroxylgroup is protected, and the 5′-hydroxyl group is protected by atemporary protecting group, with an acid.
 11. The production methodaccording to claim 10, wherein the step (1) is performed in the presenceof at least one kind of cation scavenger selected from a pyrrolederivative and an indole derivative, and further comprises a step ofneutralization with an organic base after removal of the temporaryprotecting group of the 5′-hydroxyl group.
 12. The production methodaccording to claim 9, further comprising the following step (4): (4) astep of isolating the n+p-mer oligonucleotide from the reaction mixtureobtained in step (3) by an extraction operation alone.
 13. Theproduction method according to claim 12, further comprising thefollowing step (5): (5) a step of removing all the protecting groups ofthe n+p-mer oligonucleotide obtained in step (4).
 14. The productionmethod according to claim 7, wherein the p-mer oligonucleotidecomprising a protected base, wherein the 3′-hydroxyl group isphosphoramidited, the 5′-hydroxyl group is protected by a temporaryprotecting group removable under acidic conditions, and the nucleic acidbase is protected by a group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ straight chain or branched chainalkenyl group, is the oligonucleotide comprising a protected baseaccording to any one of claims 1 to
 6. 15. The production methodaccording to claim 7, wherein the 3′-hydroxyl group of the n-meroligonucleotide is protected by a group represented by the formula(III):-L-Y—Z  (III) wherein L is a group represented by the formula (a1):

wherein * shows the bonding position to Y; ** indicates the bondingposition to a 3′-hydroxy group of the nucleotide; L₁ is an optionallysubstituted divalent C₁₋₂₂ hydrocarbon group; and L₂ is a single bond,or a group represented by **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows thebonding position to L₁, *** shows the bonding position to C═O, R¹is anoptionally substituted C₁₋₂₂ alkylene group, and R² and R³ are eachindependently a hydrogen atom or an optionally substituted C₁₋₂₂ alkylgroup, or R² and R³ are optionally joined to form an optionallysubstituted C₁₋₂₂ alkylene bond, Y is an oxygen atom or NR wherein R isa hydrogen atom, an alkyl group or an aralkyl group, and Z is a grouprepresented by the formula (a2):

wherein * shows the bonding position to Y; R⁴ is a hydrogen atom, orwhen R_(b) is a group represented by the following formula (a3), R⁴ isoptionally a single bond or —O— in combination with R⁶ to form afluorenyl group or a xanthenyl group together with ring B; Q in thenumber of k are each independently a single bond, or —O—, —S—, —OC(═O)—,—NHC(═O)— or —NH—; R⁵ in the number of k are each independently anorganic group having at least one aliphatic hydrocarbon group having oneor more branched chains and the total carbon number of not less than 14and not more than 300; k is an integer of 1 to 4; ring A optionallyfurther has, in addition to R⁴, QR⁵ in the number of k and*C(R_(a))(R_(b)), a substituent selected from the group consisting of ahalogen atom, a C₁₋₆ alkyl group optionally substituted by one or morehalogen atoms, and a C₁₋₆ alkoxy group optionally substituted by one ormore halogen atoms; R_(a) is a hydrogen atom; and R_(b) is a hydrogenatom, or a group represented by the formula (a3):

wherein * indicates a bonding position; j is an integer of 0 to 4; Q inthe number of j are each independently as defined above; R⁷ in thenumber of j are each independently an organic group having at least onealiphatic hydrocarbon group having one or more branched chains and thetotal carbon number of not less than 14 and not more than 300; R⁶ is ahydrogen atom, or optionally a single bond or —O— in combination with R⁴to form a fluorenyl group or a xanthenyl group together with ring A; andring B optionally further has, in addition to QR⁷ in the number of j andR⁶, a substituent selected from the group consisting of a halogen atom,a C₁₋₆ alkyl group optionally substituted by one or more halogen atoms,and a C₁₋₆ alkoxy group optionally substituted by one or more halogenatoms.
 16. The production method according to claim 7, wherein at leastone nucleic acid base of the n-mer oligonucleotide is protected by agroup having a C₅₋₃₀ straight chain or branched chain alkyl group and/ora C₅₋₃₀ straight chain or branched chain alkenyl group.
 17. Theproduction method according to claim 7, wherein the C₅₋₃₀ straight chainor branched chain alkyl group and/or C₅₋₃₀ straight chain or branchedchain alkenyl group are/is a C₅₋₃₀ branched chain alkyl group and/or aC₅₋₃₀ branched chain alkenyl group.
 18. A compound protected by abranched chain-containing aromatic group, which is represented by theformula (II):

wherein m is any integer of 0 or more; Base³ in the number of m+1 areeach independently an optionally protected nucleic acid base; P¹ is ahydrogen atom, or a temporary protecting group removable under acidicconditions; X is a hydrogen atom, an optionally protected hydroxylgroup, a halogen atom or an organic group crosslinked with the4-position carbon atom; X′ in the number of m are each independently ahydrogen atom, an optionally protected hydroxyl group, a halogen atom oran organic group crosslinked with the 4-position carbon atom; P² in thenumber of m are each independently a protecting group removable underbasic conditions; R³⁴ in the number of m are each independently anoxygen atom or a sulfur atom; L is a group represented by the formula(a1):

wherein * shows the bonding position to Y; ** indicates the bondingposition to a 3′-hydroxy group of the nucleotide; L₁ is an optionallysubstituted divalent C₁₋₂₂ hydrocarbon group; and L₂ is a single bond,or a group represented by **C(═O)N(R²)—R¹—N(R³)*** wherein shows thebonding position to L₁, *** shows the bonding position to C═O, R¹ is anoptionally substituted C₁₋₂₂ alkylene group, and R² and R³ are eachindependently a hydrogen atom or an optionally substituted C₁₋₂₂ alkylgroup, or R² and R³ are optionally joined to form an optionallysubstituted C₁₋₂₂ alkylene bond, Y is an oxygen atom or NR wherein R isa hydrogen atom, an alkyl group or an aralkyl group, and Z is a grouprepresented by the formula (a2):

wherein * shows the bonding position to Y; R⁴ is a hydrogen atom, orwhen R_(b) is a group represented by the following formula (a3), R⁴ isoptionally a single bond or —O— in combination with R⁶ to form afluorenyl group or a xanthenyl group together with ring B; Q in thenumber of k are each independently a single bond, or —O—, —S—, —OC(═O)—,—NHC(═O)— or —NH—; R⁵ in the number of k are each independently anorganic group having at least one aliphatic hydrocarbon group having oneor more branched chains, and the total carbon number of not less than 14and not more than 300; k is an integer of 1 to 4; ring A optionallyfurther has, in addition to R⁴, QR⁵ in the number of k and*C(R_(a))(R_(b)), a substituent selected from the group consisting of ahalogen atom, a C₁-₆ alkyl group optionally substituted by one or morehalogen atoms, and a C₁₋₆ alkoxy group optionally substituted by one ormore halogen atoms; R_(a) is a hydrogen atom; and R_(b) is a hydrogenatom, or a group represented by the formula (a3):

wherein * indicates the bonding position; j is an integer of 0 to 4; Qin the number of j are each independently as defined above; R⁷ in thenumber of j are each independently an organic group having at least onealiphatic hydrocarbon group having one or more branched chains and thetotal carbon number of not less than 14 and not more than 300; R⁶ is ahydrogen atom, or optionally a single bond or —O— in combination with R⁴to form a fluorenyl group or a xanthenyl group together with ring A; andring B optionally further has, in addition to QR⁷ in the number of j andR⁶, substituent(s) selected from the group consisting of a halogen atom,a C₁₋₆ alkyl group optionally substituted by one or more halogen atoms,and a C₁₋₆ alkoxy group optionally substituted by one or more halogenatoms.
 19. The compound according to claim 18, wherein m is
 0. 20. Thecompound according to claim 18 or 19, wherein at least one of thenucleic acid bases is protected by a group having a C₅₋₃₀ straight chainor branched chain alkyl group and/or a C₅₋₃₀ straight chain or branchedchain alkenyl group.
 21. A method of producing an n′+p′-meroligonucleotide comprising: (2′) a step of condensing a p′-meroligonucleotide, wherein p′ is any integer of one or more, wherein the3′-hydroxyl group is phosphoramidited, the 5′-hydroxyl group isprotected by a temporary protecting group removable under acidicconditions, and the nucleic acid base is optionally protected, with ann′-mer oligonucleotide, wherein n′ is any integer of one or more,wherein the 5′-hydroxyl group is not protected, and the 3′-hydroxylgroup is protected by the protecting group represented by the formula(III):L-Y—Z  (II) wherein L is a group represented by the formula (a1):

wherein * shows the bonding position to Y; ** indicates the bondingposition to a 3′-hydroxy group of the nucleotide; L₁ is an optionallysubstituted divalent C₁₋₂₂ hydrocarbon group; and L₂ is a single bond,or a group represented by **C(═O)N(R²)—R¹—N(R³)*** wherein ** shows thebonding position to L₁, *** shows the bonding position to C═O, R¹ is anoptionally substituted C₁₋₂₂ alkylene group, and R² and R³ are eachindependently a hydrogen atom or an optionally substituted C₁₋₂₂ alkylgroup, or R² and R³ are optionally joined to form an optionallysubstituted C₁₋₂₂ alkylene bond, Y is an oxygen atom or NR wherein R isa hydrogen atom, an alkyl group or an aralkyl group, and Z is a grouprepresented by the formula (a2):

wherein * shows the bonding position to Y; R⁴ is a hydrogen atom, orwhen R_(b) is a group represented by the following formula (a3), R⁴ isoptionally a single bond or —O— in combination with R⁶ to form afluorenyl group or a xanthenyl group together with ring B; Q in thenumber of k are each independently a single bond, or —O—, —S—, —OC(═0)—,—NHC(═O)— or —NH—; R⁵ in the number of k are each independently anorganic group having at least one aliphatic hydrocarbon group having oneor more branched chains and the total carbon number of not less than 14and not more than 300; k is an integer of 1 to 4; ring A optionallyfurther has, in addition to R⁴, QR⁵ in the number of k and*C(R_(a))(R_(b)), a substituent selected from the group consisting of ahalogen atom, a C₁₋₆ alkyl group optionally substituted by one or morehalogen atoms, and a C₁₋₆ alkoxy group optionally substituted by one ormore halogen atoms; R_(a) is a hydrogen atom; and R_(b) is a hydrogenatom, or a group represented by the formula (a3):

wherein * indicates the bonding position; j is an integer of 0 to 4; Qin the number of j are each independently as defined above; R⁷ in thenumber of j are each independently an organic group having at least onealiphatic hydrocarbon group having one or more branched chains and thetotal carbon number of not less than 14 and not more than 300; R⁶ is ahydrogen atom, or optionally a single bond or —O— in combination with R⁴to form a fluorenyl group or a xanthenyl group together with ring A; andring B optionally further has, in addition to QR⁷ in the number of j andR⁶, a substituent selected from the group consisting of a halogen atom,a C₁₋₆alkyl group optionally substituted by one or more halogen atoms,and a C₁₋₆ alkoxy group optionally substituted by one or more halogenatoms, by forming a phosphite triester bond via the 5′-hydroxyl groupthereof.
 22. The compound comprising a protected base according to claim1, wherein Base² in the number of q+1 are each independently a nucleicacid base protected by a group having a C₅₋₃₀ straight chain or branchedchain alkyl group and/or a C₅₋₃₀ branched chain alkenyl group.